the medicago flowering locus t homolog, …...the medicago flowering locus t homolog, mtfta1,...

The Medicago FLOWERING LOCUS T Homolog, MtFTa1,Is a Key Regulator of Flowering Time1[C][W][OA]

Rebecca E. Laurie, Payal Diwadkar, Mauren Jaudal, Lulu Zhang, Valerie Hecht, Jiangqi Wen,Million Tadege2, Kirankumar S. Mysore, Joanna Putterill, James L. Weller, and Richard C. Macknight*

Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand (R.E.L., P.D., M.J., R.C.M.);School of Biological Science, University of Auckland, Auckland 1010, New Zealand (L.Z., J.P.); School of PlantScience, University of Tasmania, Hobart, Tasmania 7001, Australia (V.H., J.L.W.); and Plant Biology, SamuelRoberts Noble Foundation, Ardmore, Oklahoma 73401 (J.W., M.T., K.S.M.)

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, wecharacterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showeddistinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) wereable to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning asflorigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions thatpromote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The keyrole MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantlylater in all conditions examined. fta1mutants do not respond to vernalization but are still responsive to LDs, indicating that theinduction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involvesother genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to thephotoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obviousflowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FTfamily.

To precisely control the timing of flowering, plantshave evolved mechanisms to integrate seasonally pre-dictable environmental cues (such as changes in pho-toperiod and prolonged periods of cold temperatures)and developmental cues (such as maturity; Amasino,2010). To allow this diversity of floral cues to influ-ence when flowering occurs in Arabidopsis (Arabidop-sis thaliana), multiple pathways converge on a smallnumber of genes, the floral integrator genes, includingthe floral promoters FLOWERING LOCUS T (FT) andTWIN SISTER OF FT (TSF; Amasino, 2010). FT andTSF are members of a family of proteins that containa phosphatidylethanolamine-binding protein (PEBP)

domain (Kardailsky et al., 1999; Kobayashi et al., 1999).In addition to the FT-like proteins, the plant PEBPfamily consists of two other phylogenetically distinctgroups of proteins: the TERMINAL FLOWER1 (TFL1)-like proteins and the MOTHER OF FT AND TFL(MFT)-like proteins (Bradley et al., 1997; Mimidaet al., 2001; Yoo et al., 2004, 2010; Yamaguchi et al.,2005). FT and TSF act redundantly to promote flower-ing under long-day (LD) photoperiods (Michaels et al.,2005; Yamaguchi et al., 2005; Jang et al., 2009). TheB-box zinc finger transcription factor CONSTANS(CO) protein induces the expression of TF and TSFin the vascular tissues under LD-inductive conditions(Kardailsky et al., 1999; Kobayashi et al., 1999; Samachet al., 2000; An et al., 2004). The LD-specific productionof CO protein is achieved through the coincidence ofthe circadian expression of CO mRNA and the stabi-lization of the CO protein in the light (Suarez-Lopezet al., 2001; Valverde et al., 2004). FT and TSF proteins,produced in the phloem (Takada and Goto, 2003;Yamaguchi et al., 2005; Jang et al., 2009), are trans-ported to the apex, where they are able to dimerizewith the bZIP transcription factor, FD, to activate theexpression of SUPPRESSOR OF OVEREXPRESSIONOF CONSTANS1 (SOC1; Michaels et al., 2005; Yooet al., 2005) and the floral meristem identity genesAPETALA1 (AP1) and LEAFY (Abe et al., 2005; Wiggeet al., 2005). Thus, FT/TSF constitute the long-soughtmobile floral signal molecule, florigen (Corbesier et al.,

1 This work was supported by the New Zealand Foundation forResearch Science and Technology (grant no. C10X0704) and by anAGMARDT Postdoctoral Fellowship (to R.E.L.).

2 Present address: Department of Plant and Soil Sciences,Oklahoma State University, Stillwater, OK 74078–6028.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Richard C. Macknight ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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2007; Jaeger and Wigge, 2007; Mathieu et al., 2007;Notaguchi et al., 2008).

It is likely that FT-like genes play a universal role inregulating flowering time. Evidence for this comesfrom experiments showing that overexpression of FThomologs causes very early flowering in eudicotplants, such as tomato (Solanum lycopersicum; Lifschitzet al., 2006), hybrid aspen trees (Populus tremula crossedwith Populus tremuloides or Populus alba; Bohleniuset al., 2006; Hsu et al., 2006), apple (Malus 3 domestica;Trankner et al., 2010), and morning glory (Ipomoea nil;Hayama et al., 2007), and monocot plants, such as rice(Oryza sativa; Izawa et al., 2002; Kojima et al., 2002) andwheat (Triticum aestivum; Yan et al., 2006). Unlike inArabidopsis, where there are just two FT-like genesthat act redundantly to regulate flowering, other spe-cies contain multiple FT-like genes and many do notappear to play a role in flowering (Chab et al., 2008;Komiya et al., 2008; Hagiwara et al., 2009; Blackmanet al., 2010; Kong et al., 2010; Kotoda et al., 2010; Pinet al., 2010). For example, there are 13 FT-like genes inrice (Chardon and Damerval, 2005), although onlytwo, RFT1 and Hd3a, have been shown to regulateflowering time (Kojima et al., 2002; Komiya et al., 2008,2009). In addition to their roles in regulating floweringtime, FT-like genes can also affect other aspects of plantdevelopment. In tomato, a balance between levels ofthe FTortholog SINGLE FLOWERTRUSS and the TFL1ortholog SELF-PRUNING (SP) determines a range ofphenotypes, including stem girth and leaf develop-ment (Shalit et al., 2009). In hybrid aspen trees (P.tremula3 P. tremuloides), an FT-like gene regulates boththe LD induction of flowering and also the short-day(SD)-induced growth cessation and bud set that occurin the autumn (Bohlenius et al., 2006). Thus, the FT-likeproteins appear to play a wider role in controllingplant development than just regulating floweringtime.

Crop and forage legumes are second only to grassesin worldwide economic importance, with floweringtime having a significant impact on production yieldsin many systems. Therefore, it is important to under-stand the molecular-genetics basis of how floweringtime is regulated in legumes. Garden pea (Pisumsativum), a LD legume, has been used in early workon the genetic control of mobile floral signals (Welleret al., 1997). More recent work in pea has shown thatLATE BLOOMER1 and DIE NEUTRALIS, orthologsof the circadian clock-associated Arabidopsis genesGIGANTEA and EARLY FLOWERING4, respectively,are required for the LD-specific production of a mobilefloral signal(s) that probably comprises at least two FT-like proteins (Hecht et al., 2007; Liew et al., 2009; Hechtet al., 2011). Five FT-like genes have been identified inpea, and these genes have different patterns of expres-sion (Hecht et al., 2011). Hecht et al. (2011) have shownthat the pea FTa1 gene corresponds to theGIGAS locus,which encodes a mobile floral signal that is essentialfor flowering under LD and promotes flowering underSD but is not required for the photoperiodic response.

They also provide evidence for a second mobile floralsignal that correlates with the expression of anotherFT-like gene, FTb2. A recent paper also implicates twoFT-like genes in the regulation of flowering of the SDlegume soybean (Glycine max; Kong et al., 2010).

Here, we characterize five FT genes from Medicago(Medicago truncatula) and show that their expressiondiffers in tissue specificity, diurnal rhythmicity, andresponse to photoperiod and vernalization. We findthat one of the FTorthologs, FTa1, plays a major role inregulating flowering time. This gene is essential for theinduction of flowering in response to vernalization butnot for the photoperiodic induction of flowering. Thiswork provides a foundation for understanding theroles of the Medicago FT family members in plantdevelopment and highlights differences between thebehavior of FT family members in Medicago and pea.

RESULTS

Medicago Has Five FT-like Genes

Our database searches for putative Medicago flow-ering-time genes revealed five FT-like genes,MtFTLa toMtFTLe (Hecht et al., 2005; Liew et al., 2009; Yeoh et al.,2011). Phylogenetic analysis of FT/TFL proteins fromother plants showed that all five Medicago FTs be-longed to the FT clade and that, in legumes, this cladeconsists of three groups of FT proteins, which we havecalled groups FTa, FTb, and FTc (Fig. 1A). The Med-icago FTs have been renamed accordingly: FTLa asFTa1, FTLb as FTa2, FTLd as FTb1, FTLe as FTb2, andFTLc as FTc (Hecht et al., 2011). TheMedicago FT genesMtFTa1, MtFTa2, and MtFTc are located next to eachother within a single bacterial artificial chromosome(BAC) clone (GenBank accession no. AC123593) map-ping to chromosome 7, and MtFTb1 and MtFTb2 arefound in tandem on another chromosome 7 BAC clone(AC127169). The FT-like genes have a similar genomicstructure to the FT genes from other species, with fourexons and three introns (Supplemental Fig. S1A), andreverse transcription (RT)-PCR confirmed that all fiveFT-like genes are expressed. An alignment of thepredicted protein sequences of the Medicago FT fam-ily is shown in Supplemental Figure S1B.

To examine how widespread the three FT groupsare, we constructed a phylogenetic tree of represen-tative FT protein sequences from the Rosid clade ofeudicot plants. Rosids consist of 17 plant orders, in-cluding the Fabales, to which the legume family(Fabaceace) belongs (AGP III, 2009). The FTa, FTb,and FTc groups were only found in the legumes andnot in any of the other Rosid orders (Fig. 1A). Whilemany plants belonging to these orders contain multi-ple FTs, these aremore related to each other than to FTswithin a different order, suggesting that they arosefrom gene duplications that occurred after the ancestorof the order evolved. The exception is Rosales, whereRcFT (from china rose [Rosa chinesis]) and FvFT (from

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woodland strawberry [Fragaria vesca]) do not groupwith the other Rosaceace family FTs (Fig. 1A). TheFabales consist of three other plant families, althoughFT sequences are only available from the Fabaceacefamily. Therefore, it is not known if the FTa, FTb, andFTc groups are present in the other Fabales families orif they are legume specific.The difference between Arabidopsis FT, which pro-

motes flowering, and TFL, which represses flowering,has been attributed to sequences within an externalloop of protein sequences that contain 14 amino acids(known as segment B) that are highly conservedwithin FTs but not in TFLs. A Gln at position 140within this region, as well as an adjacent Tyr resi-due (Tyr-85), are critical for FT function and are pre-dicted to form one wall of the ligand-binding pocket(Hanzawa et al., 2005; Ahn et al., 2006). Analysis ofthese regions from the legume FT groups and otherFTs revealed that while these sequences are highlyconserved in the other Rosid FTs, the legume FTs differfrom the consensus at three to six positions, with theFTc group proteins possessing a His rather than a Glnat the position equivalent to 140 (Fig. 1B). However, it

is not easy to predict from the segment B sequenceswhich of the Medicago FTs might function to promoteflowering.

FT Family Members Show Different Patternsof Expression

To gain insight into the potential roles of the differ-ent Medicago FT family members, we analyzed theirtranscription profiles. Total RNA was isolated from arange of tissues (Fig. 2, A and B) obtained from plantsgrown under inductive conditions (seedlings werevernalized for 14 d at 4�C and then grown underLDs [16 h of light and 8 h of dark]), and quantitative (q)RT-PCR was performed (Fig. 2C). All five FT geneswere expressed. The highest level ofMtFTa1 transcriptwas detected in the first leaf (monofoliate leaf) ofseedlings, with strong expression also observed inexpanded trifoliate leaves (but not in the immature“folded” trifoliate leaves), stem, and flowers. MtFTa1was also detected at low levels in apical bud tissues(this includes the lateral meristem and emergingleaves), flower buds and developing seed pods, roots,

Figure 1. Three classes of FT-like proteins are present in legumes (order Fabales) but not in other Rosid orders. A, Phylogram ofFT-like protein sequences from different Rosid orders. The legume FTa, FTb, and FTc groups are indicated. B, Partial amino acidalignment of FT sequences and other PEBP proteins. Asterisks on the top row indicate Tyr-85 (Y)/His-88 (H) and Gln-140 (Q)/Asp-144 (D) residues distinguishing FT-like and TFL1-like members. The black bar indicates the conserved segmental region B,corresponding to the external loop of the PEBP proteins. Sequences were aligned using ClustalW and analyzed with theGeneious software. The sequences are from Arabidopsis (At), sugar beet (Bv), orange (Citrus unshiu; Ci), pumpkin (Cucurbitamaxima; Cm), fig (Ficus carica; Fc), woodland strawberry (Fv), soybean (Gm), cotton (Gossypium hirsutum; Gh), apple (Md),Medicago (Mt), rice (Os), Lombardy poplar (Populus nigra; Pn), trifoliate orange (Poncirus trifoliata; Pt), Japanese apricot (Prunusmume; Pm), pea (Ps), Asian pear (Pyrus pyrifolia; Pp), China rose (Rosa chinensis; Rc), tomato (Le), and grape (Vitis vinifera; Vv).[See online article for color version of this figure.]

FT Regulation of Medicago Flowering

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and cotyledons (Fig. 2C). MtFTa2 was detected in alltissues examined except cotyledons. Both MtFTb1 andMtFTb2 were strongly expressed in the expandedtrifoliate leaf and monofoliate leaf but, like MtFTa1,not in the immature trifoliate leaf.MtFTb1 andMtFTb2were expressed at moderate and low levels in cotyle-dons, respectively. MtFTb1 was not detected in theother tissues examined, while MtFTb2 was expressedat very low levels in these tissues. MtFTc expressionwas detected, albeit at low levels, in apical buds, andjust detectable levels of MtFTc transcript were alsofound in stem, buds, and flowers but not in leaf orother tissues from vernalized LD plants (Fig. 2C).However, MtFTc was detected in both leaves andapical buds in SD-grown plants sampled at Zeitgebertime (ZT) 12 (the time of maximal MtFTc expression,as shown in Fig. 5Aiv below; Supplemental Fig. S2).Thus, although the five FT genes have distinct patterns

of expression, they are all expressed in the leaves, themajor site of FT/florigen production in other species(Turck et al., 2008).

We then examined if the expression of the FT geneschanges over a developmental time course, usingvernalized whole plants grown in a LD photoperiod.MtFTa1 remained relatively constant throughout de-velopment (Fig. 3A), whereas MtFTa2 and MtFTb1were expressed at the highest levels in 5-d-old plants(Fig. 3, B and C). MtFTb2 transcript levels increasedprior to flowering and then decreased about the timewhen flowers first appeared (Fig. 3D). Given thatMtFTc is expressed in apical buds, we examined itsexpression in this tissue over time (Fig. 3E). MtFTcexpression increased up to day 15, the time whenthe Medicago floral inflorescence identity gene, PRO-LIFERATING INFLORESCENCE MERISTEM (PIM;Benlloch et al., 2006), was first expressed (Fig. 3F), and

Figure 2. A and B, Expression of the FT-likegenes in different tissues of Medicago cv Jesterplants grown for 15 d (A) and 35 d (B) underLD after 2 weeks of vernalization at 4�C inthe dark. Arrowheads indicate the tissues har-vested and representative ages of plants toinvestigate expression. Harvesting was doneat 2 h after dawn (ZT2). C, Transcriptionprofiles ofMtFTa1,MtFTa2,MtFTb1,MtFTb2,and MtFTc in various tissue types as shown inA and B. The average 6 SE of three biologicalreplicates is shown for each sample, andtranscripts were normalized to PROTODER-MAL FACTOR2 (PDF2). [See online article forcolor version of this figure.]

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then it gradually decreased. We also examined the ex-pression of the FT family (excluding FTc) in leaves ofdifferent ages. Interestingly, levels ofMtFTa1 tended toincrease as the leaf aged, whereas MtFTb1 expressionclearly decreased with leaf age (Supplemental Fig. S2).Overall, our results show that, despite differences inthe developmental profile, all five of the FT genes areexpressed prior to the induction of the meristemidentify gene PIM and therefore are potential regula-tors of the floral transition.

Only MtFTa1 Is Induced Both by Vernalization and LDPhotoperiod, Conditions That Promote Flowering

Next, we investigated if the expression of any of theMedicago FT family members was induced by theenvironmental conditions that induce flowering. Flow-ering in Medicago is promoted by both exposure toprolonged periods of cold (vernalization) and LD pho-toperiods (Clarkson and Russell, 1975). Under ourconditions, vernalized Medicago cv Jester flowered 35and 56 d earlier than nonvernalized plants when grownin LD or SD photoperiods, respectively (Fig. 4A). LDs

also promoted flowering, with nonvernalized and ver-nalized plants flowering 56 and 35 d earlier, respec-tively, when grown in a LD photoperiod, comparedwith plants grown in an SD photoperiod (Fig. 4A).These differences were apparent when flowering timewas measured as either days to produce the first floweror number of nodes produced at flowering (Fig. 4A).

We then examined the effect of vernalization anddaylength on the expression of the FT family mem-bers. Comparison of FT gene expression in LD or SDphotoperiod revealed that while MtFTa1 was ex-pressed in SD conditions, it was expressed at approx-imately 2-fold higher levels in LDs (Fig. 4B). Incontrast, MtFTa2 showed the opposite response, withmoderate expression under LD and approximately7-fold higher expression under SD conditions. Theexpression of MtFTb1 and MtFTb2 was strongly influ-enced by photoperiod, with both being expressed inLDs but undetectable in SDs.MtFTa1 andMtFTa2werestrongly induced by vernalization, with neither genebeing expressed at a detectable level in LD nonver-nalized whole plants at day 15 but expressed at highlevels in LD vernalized plants (Fig. 4C; note that

Figure 3. MtFTs have different expressionpatterns during development. Relativetranscript profiles are shown over a timecourse of whole seedlings (FTa1, FTa2,FTb1, and FTb2) or the uppermost dis-sected apical buds (FTc andMtPIM). Med-icago cv Jester seedlings were vernalizedfor 2 weeks in the dark at 4�C and grownunder LDs for up to 25 d. Harvesting wasperformed at ZT2. MtPIM was used as amarker of the floral transition. A flowerbud had emerged from the node or wasvisible on whole plants harvested at 25 d(fifth or sixth node). The average 6 SE ofthree biological replicates is shown, andtranscripts were normalized to PDF2.





MtFTa1 is expressed in older nonvernalized plants [seeFig. 8E below]). In contrast, MtFTb1 and MtFTb2 wereexpressed in nonvernalized and vernalized plants(Fig. 4C). MtFTc expression was not detected usingthe RNA isolated fromwhole plants in this experimentunder any of the conditions, although it was detectedin apical buds harvested from LD- and SD-grownplants (Fig. 5Aiv).

A feature of the vernalization response is that plantsretain an epigenetic memory of the cold. To examine ifprolonged exposure to the cold directly activates theMtFTa1 or MtFTa2 gene, or if subsequent exposure toan inductive photoperiod is needed, we carried out thefollowing experiment. Medicago seeds were germi-nated, vernalized at 4�C in the dark for 14 d, and thentransferred to inductive conditions (21�C and LDphotoperiod). MtFTa1 was not expressed in germi-nated seedlings after 0, 7, or 14 d in the cold; however,after being transferred into warm LD conditions for1 d, MtFTa1 expression was just detectable, with ex-pression increasing after 7 and 14 d (Fig. 4D). Thus,vernalization per se does not induce MtFTa1 expres-sion, implying that Medicago retains a “memory”of being exposed to cold. Surprisingly, MtFTa2 re-sponded quite differently and was up-regulated after14 d at 4�C, while the seedlings were still in the coldand dark. This up-regulation of MtFTa2 expressionwas maintained after the plants were returned towarm temperatures (Fig. 4E).

In summary, the four leaf-expressed FT genes areinduced by conditions that promote flowering (MtFTa1and MtFTa2 by vernalization and MtFTa1, MtFTb1, andMtFTb2 by LD photoperiod) and therefore may playa role in the induction of flowering; however, onlyMtFTa1 is induced by both vernalization and LDphotoperiod.

Transfer from an SD to LD Photoperiod PromotesFlowering and Leads to the Rapid Up-Regulation of

MtFTa1, MtFTb1, and MtFTb2

To further investigate the effect of photoperiod onthe expression of the FT family members, we com-pared their expression over a diurnal time course fromLD- and SD-grown plants (Fig. 5A; for independentlyreplicated data, see Supplemental Fig. S3).MtFTa1wasexpressed at similar levels throughout the LD and SDdiurnal time course and did not appear to cycle in arobust manner (Fig. 5Ai). However, consistent withFigure 4B, MtFTa1 was found to be expressed at sig-nificantly higher levels in LDs compared with SDs(Fig. 5Ai). MtFTa2 was also detected at all time pointsin both the LD and SD diurnal time course and was thehighest expressed FT family member under SD con-ditions, with peak expression occurring during thedark (ZT12; Fig. 5Aii). MtFTb1 expression was notdetected at any time points of the SD diurnal timecourse but was expressed bimodally in LDs, with peak

Figure 4. Vernalization and LD photoperiod promote flowering andMtFTa1 expression. A, Flowering time of Medicago cv Jesterplants vernalized in the dark at 4�C for 2 weeks (V) or nonvernalized (NV) and grown in either LD (16 h of light/8 h of dark) or SD(8 h of light/16 h of dark) photoperiod. Flowering time was expressed as either number of days to produce the first flower (days tofirst flower) or the node fromwhich the first flower emerged (nodes to first flower). Nodes were counted from the base of the plantto the growing tip of the primary stem. The data represent average6 SE of 10 plants. B,MtFT transcript levels in whole seedlingsvernalized and grown in LD or SD. C,MtFT transcript levels in whole seedlings grown under LD, with or without vernalization.Seedlings were harvested with three nodes at 15 d old (LD) or 21 d old (SD) at ZT2. D and E, Transcription profiles of MtFTa1and MtFTa2 in seedlings grown at 4�C in the dark for 14 d and then transferred to LDs at 21�C for 14 d. The data represent anaverage 6 SE of three biological replicates, with transcripts normalized to PDF2.

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Figure 5. Photoperiodic regulation ofMtFT gene expression. A, Relative transcript levels ofMtFT genes were measured every 4 hthrough the diurnal cycle in LD and SD conditions using qRT-PCR. LDswere 16 h of light/8 h of dark and SDs were 8 h of light/16h of dark. ZT0 indicates lights on. The graphs showMtFTa1 (i),MtFTa2 (ii),MtFTb1 (iii), andMtFTc (iv) transcript levels in LD andSD, the above genes in LD (v), and the above genes in SD (vi). Below graphs v and vi, the white bars indicate day and the blackbars indicate night. Transcript abundance of the MtFT genes was normalized to TEF1a and calibrated to the sample with thehighest gene expression (FTb1 at ZT4 in LD). Total aerial parts of vernalized 10-d Jester plants grown in vitro were used. Theaverage 6 SE of qRT-PCR results from four biological replicates is shown. B, Transcript profiles of FTa1, FTa2, FTb1, and FTb2during shifts between SDs and LDs. Whole plants to be shifted were grown under SDs until the monofoliate leaf unfolded(approximately 6 d). A subset of SD plants was harvested, and the remaining plants were shifted into LDs and harvested after 1, 2,and 3 d in LD. The remaining plants were shifted back to SDs and harvested after a further 1, 2, and 3 d in SD. Seedlings wereharvested at ZT4 irrespective of LDs or SDs. The data represents an average 6 SE of three biological replicates, with transcriptsnormalized to PDF2.





expression at 4 h after dawn (ZT4) and at dusk (ZT16;Fig. 5Aiii). MtFTb2 showed a similar pattern of ex-pression (Supplemental Fig. S3). Although MtFTc wasexpressed at very low levels in whole plants, likeMtFTa2, it was also expressed at its highest level in SDsat ZT12 (Fig. 5Aiv). We were unable to detect MtFTcexpression in the leaves of LD plants; however, giventhat maximalMtFTc expression was detected in SDs atZT12, we examined whether it was expressed in theleaves at this time point. This experiment revealed thatMtFTc was expressed, albeit still at relatively lowlevels, in both the leaves and apical tissues in SDs(Supplemental Fig. S2). Overall, these results demon-strate that the different Medicago FT family membershave distinct photoperiodic responses.

To determine if any of the FT family members areregulated by photoperiod within a time frame that isconsistent with them having a role in regulatingflowering, we performed an experiment where Med-icago plants were grown under an SD photoperiodand then shifted for varying numbers of days intoinductive LD conditions. Plants grown in SD condi-tions after vernalization flowered with about ninenodes. When these plants were shifted into a LDphotoperiod when they had one visible node (andmany other nodes already formed within the shootapex), they flowered with a total of about seven nodes(Supplemental Fig. S4A). Shifting these plants from SDinto LDs for just 1 d was not sufficient to promoteflowering. However, flowering was promoted whenthe plants were shifted into LDs for 3 d or more(Supplemental Fig. S4A). This indicates that three LDswere sufficient to induce flowering in plants of thisage. Next, we examined if the expression of differentFT family members responded to changes in photo-period on a similar time scale. RNAwas obtained fromMedicago cv R108 leaves and apical buds collected atZT4 after plants were grown in SD and then trans-ferred to LDs for 1, 2, and 3 d, then back to SD for 1, 2,and 3 d (a similar experiment using RNA obtainedfrom Medicago cv Jester whole plants collected at ZT2is shown in Supplemental Fig. S4). MtFTa1 was up-regulated in both the leaves and apical buds after justone LD, and its expression increased after a further 2and 3 d in LDs (Fig. 5B). MtFTa1 returned to preshiftSD levels after just 1 d after being transferred back intoSDs (Fig. 5B). Interestingly, MtFTa2 showed the op-posite pattern of expression to MtFTa1 and was ex-pressed at lower levels in both leaves and apical budswhen grown in LD photoperiod compared with plantsshifted back into SDs (Fig. 5B). MtFTb1 and MtFTb2also responded rapidly to changes in photoperiod,with one LD being sufficient for their up-regulationand further increases seen after 2 and 3 d in LDs (Fig.5B). MtFTb1 and MtFTb2 were not detectable whenreturned to SDs (Fig. 5B). Thus, MtFTa1, MtFTb1, andMtFTb2 are up-regulated rapidly by LDs and thereforecould be responsible for the LD induction of flowering.The different behavior of MtFTa1 and MtFTa2 in theshift experiments suggests that the two genes are

regulated by different mechanisms, and this is consis-tent with their different diurnal patterns of expressionand vernalization response.

MtFTa1, MtFTb1, and MtFTc Encode FT Proteins ThatCan Complement the Arabidopsis ft-1 Mutant

To assess the potential of MtFT genes to regulateflowering time, the five Medicago genes were overex-pressed in Arabidopsis using the cauliflower mosaicvirus 35S promoter. The 35S:MtFT constructs wereintroduced into the late-floweringArabidopsis ft-1mu-tant plants (in the Landsberg erecta [Ler] background).At least 10 independent homozygous T3 lines wereproduced, and representative plants grown under LDconditions are shown in Figure 6A. 35S:MtFTa1 com-plemented the ft-1 mutation and caused plants toflower even earlier than wild-type Ler plants, with 35S:MtFTa1 plants flowering with 5.0 6 0.2 leaves com-pared with 9.1 6 0.9 leaves for Ler. In contrast, plantsoverexpressing MtFTa2 flowered with 22.4 6 0.6leaves, which was similar to ft-1 (21.4 6 2.0). MtFTb1was able to partially complement ft-1, with high-expressing lines (such as the line shown in Fig. 6)

Figure 6. Overexpression of MtFTa1, FTb1, and FTc complement theArabidopsis ft-1 mutation. A, Photograph of the 35S:MtFT lines in theArabidopsis ft-1 mutant (Ler). Ectopic expression of MtFTa1, MtFTb1,and MtFTc resulted in early flowering of ft-1 mutant plants. B, Arepresentative line expressing each MtFT construct was selected forflowering-time analysis. Total leaf number was calculated by combin-ing total rosette leaves and cauline leaves on the primary inflorescence.Data represent a minimum of 10 plants scored for each line 6 SE.MtFTa1 andMtFTc overexpression lines were consistently early flower-ing (greater than 10 independent lines scored), whereas MtFTb1transformants displayed early and intermediate flowering times (Sup-plemental Fig. S5). [See online article for color version of this figure.]

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flowering with similar numbers of leaves as Ler,whereas other lines showed intermediate floweringwith up to 15 leaves (Supplemental Fig. S5A). In con-trast, 35S:MtFTb2 lines were unable to complementft-1, even when expressed at high levels (Fig. 6; Sup-plemental Fig. S4B). 35S:MtFTc plants flowered veryearly, with some lines flowering without producingany rosette leaves and only two cauline leaves. The35S:MtFTc plants showed other abnormalities, such ascurly leaves (Supplemental Fig. S6) that have beenfound in 35S:AtFTArabidopsis plants (Teper-Bamnolkerand Samach, 2005). In summary,MtFTa1 andMtFTc canfully complement ft-1 and result in very early floweringwhen driven by the 35S promoter, whereas MtFTb1partially complements ft-1.

Overexpression of MtFTa1 Strongly AcceleratesFlowering in Transgenic Medicago Plants

Medicago cv R108 was transformed with the same35S:MtFTa1 construct that was used to complementthe Arabidopsis ft-1 mutant. As controls, we trans-formed plants with a 35S:GUS construct and also re-generated Medicago plants from leaf explants thathad not been infected with Agrobacterium tumefaciens.The flowering time of T0 plants (plants regenerateddirectly from explants) was measured. Nine indepen-dently generated 35S:MtFTa1 plants were obtained,and all flowered significantly earlier than the 35S:GUSand regenerated plant controls (Fig. 7, A and C). Innonvernalized plants, MtFTa1 was expressed at ap-proximately 30- to 3,400-fold higher levels in the three35S:MtFTa1 lines examined compared with the 35S:GUS or regenerated control plants (Fig. 7B). We thenmeasured the flowering time of T1 progeny from twoof the 35S:MtFTa1 lines grown in LD photoperiodwithout vernalization (Fig. 7, D and E). All 12 progenyfrom the first line, M1-4, flowered significantly earlier(average, 28.6 6 0.9 d) than R108 (average, 54.1 61.1 d). The second line segregated early-floweringplants that flowered after just 24 6 0 d with only twonodes and a late-flowering plant that flowered after 57d with 11 nodes. Thus, MtFTa1 can induce floweringwhen overexpressed in Medicago, even in the absenceof vernalization, indicating that FT expression is alimiting factor in promoting the floral transition. The35S:MtFTa1 plants also exhibited increased internodeelongation and less branching compared with the 35S:GUS and regenerated control plants, resulting in amore erect phenotype, indicating that, in addition topromoting flowering, MtFTa1 influences other aspectsof plant development.

Medicago fta1 Mutants Are Late Flowering

The fact thatMtFTa1 is expressed in the leaves, is up-regulated by conditions that promote flowering (ver-nalization and LDs), and causes early flowering whenoverexpressed in Arabidopsis and Medicago stronglyimplicates MtFTa1 as a key regulator of flowering. To

provide more direct evidence for the role of MtFTa1in flowering, we set out to identify plants carryingmutations in this gene. Using Medicago cv R108Tnt1 retrotransposon-tagged insertion mutants (Tadegeet al., 2008), a reverse genetic approach was employedto screen for Tnt1 insertions within the MtFTa1 geneusing PCR. Two different lines were found to haveTnt1 sequences within the MtFTa1 gene. Line NF3307contained an insertion within exon 1 (fta1-1), and Tnt1was inserted within intron 1 in line NF2519 (fta1-2; Fig.8A). Both lines segregated plants with a late-floweringphenotype (fta1-1 is shown Fig. 8B and fta1-2 is shownin Supplemental Fig. S7). No RT-PCR products weredetected using primers across the insertion sites, indi-cating that the Tnt1 sequences prevent a correctlyspliced mRNA from being produced (Fig. 8C). Next,we investigated if late flowering correlated with ho-mozygous Tnt1 insertions. In both lines, approxi-mately 25% of the plants were late flowering, and allwere homozygous for the Tnt1 insertion within theMtFTa1 gene (Supplemental Fig. S7). Together, theseresults indicate that disrupting the MtFTa1 gene re-sults in a late-flowering phenotype. Loss of FTa1 alsoaffected the growth habit (Fig. 8B). In comparison withthe wild type, the fta1 mutant showed dramaticallyreduced elongation of main stem internodes, moreprofuse and vigorous lateral branching at lower nodes,and a more prostrate habit overall.

To investigate the environmental conditions in whichMtFTa1 functions to promote flowering, wild-type R108(from NF3307) and the fta1-1 mutant were either ver-nalized for 2 weeks at 4�C or not vernalized and grownin an LD or SD photoperiod. The vernalized fta1-1 mu-tant flowered later than the wild type when grown inboth LD and SD photoperiods, consistent with theexpression data showing that while MtFTa1 is up-regulated by the LD photoperiod, it is also expressedin SD conditions (Figs. 4C and 5). However, thefta1-1 mutant still flowered significantly earlier whengrown in LD compared with SD photoperiods (92 dcompared with 141 d; Fig. 8D). Similarly, the non-vernalized fta1-1 mutant also flowered earlier in LDcompared with SD conditions (94 d compared with144 d; Fig. 8D). Therefore, while theMtFTa1 gene playsa major role in the promotion of flowering under theLD photoperiod, an MtFTa1-independent pathwaymust also exist to allow Medicago to flower earlier inLD conditions.

In both LD and SD conditions, the vernalized fta1-1mutant flowered at the same time as the nonvernal-ized fta1-1 mutant (Fig. 8D). Thus, the fta1-1 mutantis completely unresponsive to vernalization, indi-cating that MtFTa1 is the major target of the ver-nalization pathway. Given that we did not detectMtFTa1 expression in nonvernalized 15-d-old plants,it was surprising that the fta1-1 mutant floweredlater than wild-type plants without vernalization.Therefore, we examined MtFTa1 expression in oldernonvernalized plants and found that MtFTa1 wasindeed expressed in plants prior to flowering (Fig.





8E). Thus, the expression of MtFTa1 in nonvernalizedplants later in development is sufficient to promoteflowering.

Altered Expression of FT Family Members in fta1

To investigate ifMtFTa1 affects the expression of anyother FT genes, we first examined the expression ofMtFTa2, MtFTb1, and MtFTb2 in fta1-1 mutant plantsgrown in SD and shifted to LD photoperiod. Thisexperiment was performed at the same time as the

shift experiment with wild-type plants shown in Fig-ure 5B. The fact that MtFTa1 is expressed at higherlevels in LD than in SD photoperiod, whereas MtFTa2shows the opposite expression pattern (Fig. 5), raisedthe possibility that MtFTa1 might repress MtFTa2expression. However, MtFTa2 was expressed in boththe leaves and nodes in fta1-1 mutant plants and atsimilar levels to those in wild-type plants. Therefore,the reduced levels of MtFTa1 in SDs are not responsi-ble for the rapid up-regulation of MtFTa2 that occurswhen plants are shifted back to SDs. Similarly, it

Figure 7. Overexpression of FTa1 in Medicago results in early flowering. A, Flowering time of Medicago cv R108 transformedwith 35S:FTa1 or 35S:GUS. Regeneration controls (RC) are plants that were regenerated from leaf explants in the absence ofphosphinothricin selection. The graph shows flowering times of T0 transformants and control plants in LD conditions withoutvernalization, expressed as nodes to first flower. The data from nine independent 35S:FTa1 transformants, five independent 35S:GUS lines, and eight RC plants were used to calculate the mean number of nodes to flowering 6 SE. B, MtFTa1 transcriptaccumulation in T0 transformants and control plants was measured in LD conditions using qRT-PCR. Relative transcriptabundance ofMtFTa1 in fully expanded trifoliate leaves at ZT1.5 is shown for three independent 35S:FTa1 plants, one 35S:GUSplant, and one RC plant. Data represent average 6 SE of two biological replicates, with transcripts normalized to TEF1a. C, T0transformant and control plants grown under LDs. Photographs were taken 42 to 44 d after transfer of regenerated plantlets tosoil. The white arrowhead indicates a flower. D and E, Graphs showing the days to first flower (D) and nodes to first flower (E) of35S:FTa1 T1 and R108 control plants in LD conditions without vernalization. The flowering time of the progeny of twoindependent T0 transformants was measured. All the progeny of M1-4 flowered early (n = 12), while the progeny of M18-2showed segregation, with two plants flowering early and one plant flowering as late as the R108 control plants (n = 10). [Seeonline article for color version of this figure.]

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was possible that the up-regulation of MtFTa1 inLDs might contribute to the expression of MtFTb1and MtFTb2 only under LDs. However, both MtFTb1and MtFTb2 were up-regulated upon transfer fromSD to LD conditions in the fta1-1 mutant, indicatingthat FTa1 is not required for their expression in LDs(Fig. 9B).

To further investigate the regulation of the FT genes,we examined their expression in wild-type and fta1-1mutant plants in leaves and apical bud tissue atdifferent stages of development. RNA was isolatedfrom plants with two nodes (approximately 10 d), fivenodes (approximately 20 d), and plants that hadflowered (approximately 60 d for wild-type plantsand approximately 80 d for the late-flowering fta1-1mutant). As expected, MtFTa1 was expressed in bothleaves and apical buds at these time points but was notdetected in the fta1-1 mutant (Supplemental Fig. S8A).Prior to flowering, MtFTa2 was comparable betweenthe wild type and fta1-1 in both leaves and apical buds(P = 0.45 and P = 0.57, respectively; Fig. 9A). However,after flowering, MtFTa2 was expressed at significantlyhigher levels (P , 0.01) in the fta1-1 mutant comparedwith the wild type (approximately 10-fold and ap-proximately 8-fold higher in leaves and apical buds,respectively; Supplemental Fig. S8B). MtFTb1 wasexpressed at slightly higher levels in the leaves of thefta1-1 mutant compared with the wild type, althoughthis difference was not statistically significant (P =0.063). However, after flowering, MtFTb1 was ex-pressed at significantly higher levels in fta1 comparedwith the wild type (P = 0.014; Supplemental Fig. S8C).In contrast, there were no significant differences inMtFTb2 between the fta1-1 mutant and the wild type(P = 0.677; Fig. 9B). The increased expression ofMtFTa2 and MtFTb2 in the fta1-1 mutant might bedue to the significant difference in the age of the fta1-1and wild-type plants when they flowered, althoughit might also be due to MtFTa1 directly or indirectlysuppressing MtFTa2 and MtFTb1 expression. Addi-tional experiments will be required to establish whyMtFTa2 and MtFTb1 are expressed at higher levels inthe fta1-1 mutant. However, the effect of the fta1-1mutant onMtFTc expression was more dramatic. Priorto flowering, at the two- and five-node stages, MtFTcwas only expressed in the apical buds of wild-typeplants and could not be detected in the fta1-1 mutant(Fig. 9C). However, MtFTc expression was observedonce fta1-1 plants had flowered (Fig. 9C). The fact thatin LDs,MtFTc is only expressed in the apical buds andonly once the fta1-1 plants flowered suggests thatMtFTc might be expressed in a similar manner to thefloral meristem identity genes. We examined the ex-pression of MtPIM and a Medicago homolog of FRUIT-FULL, FULc (Hecht et al., 2005). Both of these geneswerenot expressed in wild-type plants at the two-node stage(approximately 10 d after germination), whereas MtFTcwas detected at this stage (Fig. 9C). Thus, MtFTc isexpressed prior to the induction of the floral meristemidentity genes.

Figure 8. fta1 mutants are late flowering. A, Schematic of the MtFTa1gene with the positions of fta1-1 (NF3307; +56) and fta1-2 (NF2519;+273) marked. Exons are shown by black boxes, and thin linesrepresent introns. B, Photograph of R108 (wild type [WT]) and anfta1-1 mutant plant taken 35 d after germination. R108 is producingflowers (arrowheads). Plants were vernalized for 2 weeks and thengrown in LDs. C, A graph showing the relative expression ofMtFTa1 inR108 and plants homozygous for the fta1-1or fta1-2mutation. RNAwasextracted from fully expanded trifoliate leaves grown under LDs at 15 dafter germination, following a vernalization treatment. Data representaverage relative expression for three biological replicates 6 SE. D, Acomparison of flowering times (measured using days to first flower) ofR108 and fta1-1 plants in different growth conditions (LDs or SDs),vernalized (V) or nonvernalized (NV). Data represent average of at leastsix plants 6 SE. E, The relative expression of MtFTa1 in R108 grown inLDs (nonvernalized). The uppermost fully expanded trifoliate leaf washarvested from plants with four, six to eight, 14 to 16, and 18+ nodes.Plants with 18+ nodes had flowered. Data represent average for threebiological replicates 6 SE. [See online article for color version of thisfigure.]





ftc Mutants Have No Obvious Phenotype

MtFTc is expressed in apical bud tissue prior to theexpression of the floral meristem identity genes, andits expression is delayed in the fta1-1 mutant, suggest-ing that it might be involved in the floral transition. Toinvestigate the role of MtFTc, we screened the Tnt1lines for inserts within the MtFTc gene using the sameapproach we used to identify the fta1 mutants. Threeindependent Tnt1 insertions were identified in MtFTcwithin the 5# untranslated region (NF6335; ftc-1), exon1 (NF4345; ftc-2), and intron 1 (NF4913; ftc-3; Fig. 10A).The Tnt1 insertions all segregated approximately 3:1,and homozygous lines for each of the lines were iden-tified (Supplemental Fig. S9). qRT-PCR using primersto amplify across MtFTc exons 1 and 2 revealed thatMtFTc is expressed at lower levels in ftc-1 homozygous

plants and that, in ftc-3 homozygous plants, a low levelof correctly spliced transcript persists despite thelarge Tnt1 insert. In contrast, MtFTc was not detect-able in ftc-2mutant plants, indicating that only ftc-2 islikely to be a null mutant (Fig. 10B). Homozygousplants for each of the three Tnt1 insertions developednormally and were phenotypically indistinguishablefrom the wild type, with normal flowers forming atapproximately the same time (Fig. 10C; SupplementalFig. S9). Thus, MtFTc does not appear to play a majorrole in regulating Medicago flowering in LDs, con-sistent with its confined expression pattern in apicalbuds. However, given that MtFTc is also expressed inthe leaves in SD-grown plants (Supplemental Fig. S2),we are currently investigating if MtFTc influencesflowering in SDs.

Figure 9. Altered expression of Medicago FTs and floral meristem identify genes in the fta1-1mutant. A and B, Relative transcriptlevels of FTs in wild-type (black or gray) and fta1-1 (white) leaves or dissected apical buds during shifts from SDs and LDs, asdescribed in Figure 5. C, Relative transcript levels of FTc and themeristem identify genes,MtPIM andMtFULc, in dissected apicalbud tissue from wild-type R108 and fta1-1 plants approximately 10 d old with two nodes (2n), approximately 25 d old with fivenodes (5n), or once the plants had flowered. Plants were vernalized for 2 weeks in the dark at 4�C and grown under LDs. Tissuewas harvested at ZT2. The data represent an average 6 SE of three biological replicates, with transcripts normalized to PDF2.

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DISCUSSION

FT-like proteins appear to play a universal role inregulating flowering time (Turck et al., 2008). Here, wehave characterized five Medicago FT family membersand show that the different FTs have distinct patternsof expression and responses to environmental cues.We demonstrate that only one of the FT genes,MtFTa1,is regulated by both vernalization and a LD photope-riod and that this gene plays a major role in theregulation of Medicago flowering.Unlike other Rosids, the legumes have three distinct

groups of FT proteins, FTa, FTb, and FTc (Hecht et al.,2011; Fig. 1A). MtFTa1 encodes an FTa group protein,and recent work by the Weller group has shown thatthe pea FTa group gene, PsFTa1, also plays a key role inregulating flowering time. PsFTa1 corresponds to theGIGAS locus, is essential for flowering under LDconditions, and promotes flowering under SD condi-tions (Hecht et al., 2011). Like Medicago, pea has atleast five FT-like genes, and these genes have showndistinct patterns of expression. The pea FTb groupgene, PsFTb2, is also implicated in the LD induction offlowering (Hecht et al., 2011). Ten FT genes have beenfound in soybean, a SD legume (Kong et al., 2010;Hecht et al., 2011). Two of these soybean FT genes(GmFT2a and GmFT5a) have recently been implicatedin controlling flowering (Kong et al., 2010). GmFT2aencodes an FTa group protein and GmFT5a encodes anFTc group protein, named GmFTa3 and GmFTc1, re-spectively, by Hecht et al. (2011; Fig. 1A). Therefore, itappears that flowering in legumes involves multipleFT genes that belong to different FT groups.Overexpression of the different Medicago and pea

FT family members in the Arabidopsis ft-1 mutantrevealed that not all the FT-like genes are functional,and even those that were able to complement ft-1varied in the degree to which they promoted flower-ing. Therefore, sequence variation within the FT-likefamily members affects their ability to promote flower-ing. Conserved sequences within the FT proteins arerequired for the promotion of flowering. The Tyr-85/

His-88 and Gln-140/Asp-144 residues, as well as se-quences within the external loop of the PEBP proteins,known as segment B, determine whether the FT-likeand TFL1-like members promote or repress flowering(Hanzawa et al., 2005; Ahn et al., 2006). While the alllegume FT-like proteins have the conserved Tyr-85, thelegume FTc group proteins, which strongly promoteflowering, have a His rather than a Gln at position 140.Segment B, which is conserved in the FT-like proteinsfrom other plants, is more divergent in the legume FTs.Amino acid changes within this region can have aprofound effect on the activity of the FT-like proteins. Insugar beet (Beta vulgaris), just three amino acid changeswithin this region (Tyr-138/Asn-134, Gly-141/Gln-137,and Trp-142/Gln-138) determine the ability of BvFT2and BvFT1 to promote and repress flowering, respec-tively (Pin et al., 2010). These three amino acids are con-served within strongly activating MtFTa1 and PsFTa1/GIGAS proteins. However, interestingly, two of theseamino acids (equivalent to Tyr-138 and Gly-141 inBvFT2) vary in the FTc proteins. Tyr-138 is replacedwith other polar amino acids (Phe or Ile), whereas inBvFT1, it is replaced with a nonpolar amino acid, Asn.This suggests that while conservative substitutions atTyr-138 do not abolish FT’s ability to promote flowering,nonconservative changes might have a more profoundeffect. In contrast, changes at Gly-141, a nonpolar aminoacid, are probably less important, as the acidic residueAsp or Glu is present at the equivalent position inFTc proteins. Thus, it is unlikely that the presence ofthe polar amino acid Gln at this position in BvFT1 isresponsible for the late-flowering properties of BvFT1.All the FTs except BvFT1 have a Trp at the equivalentposition to 142 in BvFT2. It is likely, therefore, that thisamino acid change, probably together with the Try-138/Asn change, is key to the BvFT1 repressive func-tion. However, it is not clear whether variation in theamino acids within the B segment of the legume FTs isresponsible for their differing abilities to promote flow-ering. Clearly, variation within other regions also affectthe ability of the different FTs to promote Arabidopsis

Figure 10. Wild-type flowering of ftcmutants caused by Tnt1 insertions. A,Schematic showing the positions ofTnt1 insertions in the MtFTc gene.Using a reverse genetics approach,Tnt1 insertions were identified in the5# untranslated region (NF6335; ftc1),exon 1 (NF4345; ftc2), and intron1 (NF4913; ftc3). B, Relative expres-sion ofMtFTc in apical buds from wildtype (WT) and three ftcmutant lines. C,Flowering time ftc mutants in vernal-ized LD plants. Data represent theaverage of eight plants homozygousfor the Tnt1 insertion 6 SE.





flowering when overexpressed, as MtFTa1/MtFTa2 andMtFTb1/MtFTb2 have identical B segments, althoughonly MtFTa1 and MtFTb1 cause early flowering.

Our results indicate that MtFTa1 plays a key role inthe promotion of flowering by vernalization. The ex-pression of MtFTa1 is strongly induced by a vernaliza-tion treatment (Fig. 4C), and the fta1-1 mutant appearsto be unable to respond to vernalization (Fig. 8D),indicating that MtFTa1 is the major, and possibly only,target of the vernalization pathway. Although anotherFT gene, MtFTa2, is also induced by vernalization, thisgene was unable to complement the ft-1mutationwhenoverexpressed in Arabidopsis. Therefore, it appearsthat MtFTa2 does not encode a protein capable of in-ducing flowering. The identification and characteriza-tion of an MtFTa2 mutant will be required to establishif it plays any role in Medicago flowering.

A hallmark of the vernalization process is that plantsretain an epigenetic memory of being exposed to thecold. To investigate if up-regulation of the MtFTa1 orMtFTa2 gene in response to vernalization occurs onlyafter the plants are returned to warm temperatures, weexamined their expression in germinated seedlingsgrowing at 4�C in the dark and then growing at 21�Cin LDs. MtFTa1 was not expressed in seedlings duringthe cold but was up-regulated when the plants weretransferred into warm LDs (Fig. 4D). This impliesthat, as in other plants, Medicago plants retain a mem-ory of the cold. Interestingly, MtFTa2 expression wasup-regulated while still in the cold; however, its ex-pression was maintained when the plants were grownin warm temperatures, suggesting that there might alsobe a epigenetic basis to its regulation.

Although MtFTa1 is strongly induced by vernaliza-tion,MtFTa1 is expressed at low levels in nonvernalizedplants (Fig. 8E) and this low level of expression issufficient to promote flowering, as the nonvernalizedfta1-1mutant flowered significantly later than wild-typeplants (Fig. 8D). Thus, although MtFTa1 is the majortarget of the vernalization pathway, it also plays animportant role in determining the flowering time ofnonvernalized plants. In vernalization-responsive Arab-idopsis accessions, FT expression is repressed prior tovernalization by FLOWERING LOCUS C (FLC) inter-acting with the FT promoter (Searle et al., 2006). Vernal-ization results in the epigenetic silencing of the FLCgene, which then allows the FT gene to be up-regulatedby the photoperiod pathway. Medicago does not pos-sess an obvious FLC homolog (Hecht et al., 2005), raisingthe possibility that the vernalization-responsive regula-tion of MtFTa1 might stem from a different mechanismto that of Arabidopsis. This may not be surprising, as ithas been suggested that since flowering plants evolvedin a warm environment where vernalization would notbe necessary, the cold induction of floweringmight haveevolved independently in different families of floweringplants (Amasino, 2010). In support of this, a recent studyhas shown that vernalization of cultivated sugar beet, aeudicot in the family Amaranthaceae, involves a differ-ent mechanism from Arabidopsis, a member of the

Brassicaceae (Pin et al., 2010). In sugar beet, ratherthan silencing an FLC-like gene, vernalization down-regulates an FT gene, BvFT1, that has evolved the abilityto repress the expression of another FT gene, BvFT2,shown to be essential for flowering (Pin et al., 2010).None of the Medicago FT genes are down-regulatedby vernalization. Therefore, the up-regulation ofMtFTa1by vernalization does not appear to involve anotherFT. Vernalization in cereals also involves the down-regulation of an FT repressor, in this case the CO,CO-like, and TIMINGOF CAB EXPRESSION1 domaintranscription factor VRN2 (Yan et al., 2004; Hemminget al., 2008). We are currently searching for genes thatencode vernalization-responsive repressors of MtFTa1expression.

The expression of MtFTa1 is induced by a LDphotoperiod (Fig. 5B), and consistent with a role inpromoting flowering in this condition, the fta1 mutanthas significantly delayed flowering in LDs comparedwith wild-type plants (Fig. 8D). However, the fta1mutant is still capable of responding to photoperiod,indicating that another pathway exists. A candidatefor this pathway isMtFTb1. Consistent with a potentialrole as a florigen, MtFTb1 is expressed in the leavesunder LD photoperiods but is not expressed at de-tectable levels in SD photoperiods (Fig. 5B) and cancomplement the Arabidopsis ft-1mutant when overex-pressed at high levels (Fig. 6). Unfortunately, we havenot yet identified a Tnt1 insertion within the MtFTb1gene to examine whether it is involved in regulatingflowering. However, recent work in pea supports theidea that photoperiodic regulation of flowering intemperate legumes might involve multiple FT genes(Hecht et al., 2011). Hecht et al. (2011) have shown thatPsFTa1 corresponds to the GIGAS gene. The Psfta1/gigasmutant is deficient in a mobile floral signal that isrequired for flowering under LD and promotes flower-ing under SD conditions; however, the mutant can stillrespond to photoperiod (Beveridge and Murfet, 1996;Hecht et al., 2011). Hecht et al. (2011) provided evi-dence that the photoperiodic induction of floweringinvolves a second mobile floral signal that correlateswith the expression of the FTb group gene, PsFTb2,which, like the Medicago FTb1 and FTb2 genes, isinduced by LDs. Thus, the photoperiodic induction offlowering of bothMedicago and peamight involve FTaand FTb group FT proteins. Interestingly, MtFTa1 andMtFTb1 are regulated differently by photoperiod; forexample, MtFTa1 lacks a robust diurnal rhythm and isexpressed in SDs, whereas MtFTb1 is regulated diur-nally and not expressed in SDs. This suggests that twomechanisms might exist to mediate the LD inductionof the FTs.

Although the FTa group genes MtFTa1 and PsFTa1/GIGAS play important roles in the regulation ofMedicago and pea flowering, respectively, they donot have exactly the same function. While flowering ofthe Medicago fta1 mutant is delayed under LDs, nor-mal flowers are eventually produced and themeristemidentity genes MtPIM and MtFULc are expressed in

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the apical buds at flowering (Fig. 9C). In contrast,Psfta1/gigas mutants generally fail to produce flowersunder LDs, and this is associated with a lack ofexpression of the floral meristem identity genes PIMand SEPALLALA1 and the reduced expression ofUNIFOLIATA (Hecht et al., 2011). Interestingly, flower-ing of Psfta1/gigas is only delayed under the SD pho-toperiod, and normal flowers are produced (Beveridgeand Murfet, 1996; Hecht et al., 2011). The Medicagofta1mutant also has delayed flowering under SDs. Therole ofMtFTa1 in the induction of flowering under SDsis in contrast to Arabidopsis, where FT/TSF play a veryminor role in SDs, with the ft-10 tsf-1 double mutantflowering only slightly later than wild-type plants inSDs (Jang et al., 2009). Instead, the SD floweringof Arabidopsis involves the FT/TSF-independent in-duction of SOC1 by GA (Borner et al., 2000; Janget al., 2009). Recently, two FT genes (GmFT2a/FTa3 andGmFT5a/FTc1) have been implicated in the floweringof the SD legume soybean. These genes are expressedat low levels in LDs and up-regulated under SDs.Thus, the altered regulation of the FT genes appears tobe a key difference between LD and SD legumes.In addition to the LD-induced MtFTa1 and MtFTb1

genes, MtFTc was also able to complement the Arabi-dopsis ft-1 mutant. Although more divergent in se-quence compared with the other Medicago FTs, MtFTccaused Arabidopsis to flower very early when over-expressed. This suggested thatMtFTcmight play a rolein controlling flowering time. However, the ftc mu-tants flowered at the same time as the wild type anddid not have any obvious phenotypes. Thus, the role ofMtFTc is not clear. A possible explanation for the lackof a flowering-time phenotype in the ftc mutant is thatMtFTa1 and MtFTc have overlapping or partially re-dundant roles and that MtFTa1 activity can compen-sate for the lack of MtFTc. However, the fta1/ftc doublemutant needed to address this question will be diffi-cult to obtain, as the two genes are tightly linked (onlyapproximately 30 kb apart). MtFTc is expressed in theapical buds but not in leaves, and its expression isassociated with flowering. The fta1 mutant has nodetectable MtFTc expression prior to flowering; how-ever, MtFTc is expressed once fta1 plants flower. Thismay be similar to the Arabidopsis floral meristemidentity gene AP1, which is activated by FT but canalso be activated by other pathways that induceflowering. However, MtFTc expression was detectedprior to the induction of theMedicago ortholog ofAP1,MtPIM, and the meristem identity geneMtFULc. Thus,MtFTc expression appears to be associated with thefloral transition rather than simply with the formationof flowers. In pea, the PsFTc gene is also expressed inapical buds but not in leaves (Hecht et al., 2011). LikeMedicago, the PsFTc gene is up-regulated at a similartime to the pea meristem identity genes, and itsexpression is reduced in the Psfta1/gigasmutant (Hechtet al., 2011). In contrast to Medicago and pea, thesoybean FTc group gene GmFT5a/FTc1 is thought toplay a role in regulating flowering, as it is up-regulated

by inductive SD photoperiods and is expressed in theleaves. Thus, the FTc gene may play a different role inthe tropical SD legume, soybean, compared with thetemperate LD legumes, Medicago and pea.

In pea, the expression of the FTa2 gene is abolished inthe fta1/gigas mutant, indicating that FTa1 directly orindirectly up-regulates FTa2 expression. However, this isnot the case in Medicago, where lower expression ofFTa2 is not seen in the fta1 mutant (Fig. 5). If anything,FTa1might lead to the down-regulation of FTa2, as oncethe plants flower, FTa2 is expressed at higher levels inthe fta1 mutant compared with wild-type plants (Sup-plemental Fig. S8). Other differences also exits betweenthe pea and Medicago FTa2 genes. PsFTa2 can comple-ment Arabidopsis ft-1 (Hecht et al., 2011), whereasMtFTa2 was unable to complement the Arabidopsisft-1 mutation and therefore probably does not encode aprotein capable of affecting flowering. MtFTa2 expres-sion is induced by vernalization and SDs, environmen-tal conditions that might allow Medicago to adapt itsflowering response to suit particular regions. Therefore,more recent mutations within MtFTa2 might have re-sulted in the loss of its ability to function as a florigen. Itis also possible thatMtFTa2 actually delays flowering inSDs, perhaps by competing with MtFTa1. Further ex-periments will be needed to establish if MtFTa2 plays arole in flowering. Similarly, while MtFTb2, like MtFTb1,is only expressed in LD photoperiods, MtFTb2 cannotcomplement Arabidopsis ft-1, while MtFTb1 partiallycomplements ft-1. Interestingly, in pea, both the PsFTb1and PsFTb2 genes can complement Arabidopsis ft-1;however, only PsFTb2 is induced by LDs and PsFTb1is expressed at very low levels in both LD and SDthroughout development (Hecht et al., 2011). Thus, inboth Medicago and pea, probably only one FTb groupgene functions in the LD induction of flowering. Se-quence variants within FT homologs from sunflower(Helianthus annuus; Blackman et al., 2010), wheat (Yanet al., 2006), rice (Hagiwara et al., 2009), andArabidopsis(Schwartz et al., 2009) have been shown to be importantfor flowering-time adaptation. Since there is significantvariation in flowering time in different Medicago acces-sions (Skinner et al., 1999), it is possible that variation inthe activity or expression of the various Medicago FTfamily members underlies some of this variation.

In conclusion, this work provides a detailed char-acterization of the Medicago FT gene family anddemonstrates that MtFTa1 plays a key role in theregulation of flowering. Since the other FT familymembers are also regulated by vernalization and/ordaylength, it possible that they also influence flower-ing. Future work will focus on understanding the rolesof the different FT family members.

MATERIALS AND METHODS

Bioinformatics

Medicago (Medicago truncatula) homologs of Arabidopsis (Arabidopsis

thaliana) FT were identified using tBLASTn searches against Medicago ESTs





and BAC sequences. Alignments were performed and aligned using ClustalW

in the Geneious software package. Phylogenetic relationships among sequences

were estimated using the neighbor-joining method in the MEGA 4.0.2 program

(Kumar et al., 2008). The accession numbers for sequences are as follows:

Arabidopsis AtFT (NM_105222), AtTSF (NM_118156), AtTFL (NM_120465),

AtBFT (NM_125597), AtMFT (NM_101672); sugar beet (Beta vulgaris) BvFT1

(HM448910), BvFT2 (HM448912); orange (Citrus unshiu) CiFT (AB027456), CiFT2

(AB301934), CiFT3 (AB301935), CmFT-like2 (ABI94606); pumpkin (Cucurbita

maxima) CmFT-like1 (ABI94605); fig (Ficus carica) FcFT (BAI60052); woodland

strawberry (Fragaria vesca) FvFT (CBY25183); soybean (Glycine max) GmFTa1

(AB550124; Glyma16g04840), GmFTa2 (AB550125; Glyma19g28390), GmFTa3/

FT2a (AB550122; Glyma16g26660), GmFTa4 (Glyma16g26690), GmFTb1

(Glyma08g47820), GmFTb2 (Glyma08g47810), GmFTb3 (AB550120; Glyma18-

g53680), GmFTb4 (AB550121; Glyma18g53690), GmFTc1/5a (AB550126; Gly-

ma16g04830), GmFTc2 (Glyma19g28400); cotton (Gossypium hirsutum) GhFT

(HM631972); apple (Malus 3 domestica) MdFT1 (AB161112), MdFT2

(AB458504); Medicago MtFTa1 (HQ721813), MtFTa2 (HQ721814), MtFTb1

(HQ721815),MtFTb2 (HQ721816),MtFTc (HQ721817),MtTFL1 (Medtr7g127250),

MtBFT (AC146807_6.1), MtMFT (Medtr4g155400); rice (Oryza sativa) OsHd3a

(NM_001063395); Lombardy poplar (Populus nigra) PnFT1 (AB106111), PnFT2

(AB109804), PnFT3/4 (AB110612); trifoliate orange (Poncirus trifoliate) PtFT

(EU400602); Japanese apricot (Prunus mume) PmFT (BAH82787); pea (Pisum

sativum) PsFTa1 (HQ538822), PsFTa2 (HQ538823), PsFTb1 (HQ538824), PsFTb2

(HQ538825), PsFTc (HQ538826); Asian pear (Pyrus pyrifolia) PyFT (BAJ11577);

China rose (Rosa chinensis) RcFT (CBY25182); tomato (Solanum lycopersicum)

LeSP3D (AY186735); and grape (Vitis vinifera) VvFT-like (DQ871590).

Plant Materials and Growth Conditions

Medicago cv Jester was kindly supplied by Seedmark, accession R108

(Hoffmann et al., 1997) and various Tnt1 insertion lines were obtained from

the Noble Foundation, and the R108-1 (c3) line used for transformation was

from Pascal Ratet (Centre National de la Recherche Scientifique, Gif-sur-

Yvette, France). Medicago and Arabidopsis plants were grown under the

following regimes: 16 h of light/8 h of dark for LDs and 8 h of light/16 h of

dark for SDs, in growth cabinets maintained at 21�C with 30% to 40%

humidity, and a light intensity of approximately 115 mmol m22 s21. For

vernalization treatment, Medicago seeds were first scarified, germinated

overnight at 21�C on moist petri dishes, and placed at 4�C in the dark for 2

weeks. For cultivation of Medicago plants, soil was composed of a mixture of

nine parts Black Magic seed-raising mix (Yates Orica New Zealand), three

parts coarse-graded vermiculite (Pacific Growers Supplies), and one part No.

2 Propagating Sand (Daltons). After 1 month of growth, watering was sup-

plemented with hydroponics medium (without Na2SiO3; Gibeaut et al., 1997).

For in vitro culture, Medicago cv Jester or R108 seeds were scarified, sterilized,

and cold treated for 3 d or 2 weeks (vernalized) at 4�C. Germinated seedlings

were transferred to half-strength SH9 medium (Cosson et al., 2006) with HP

696-7470 agar (Kalys) in Magenta boxes (Sigma). Flowering time of Medicago

plants was recorded as the number of days from germination to flowering

(when the first fully opened flower was observed) and/or the number of

nodes on the main axis at flowering.

Arabidopsis Complementation

PCR-amplified MtFT DNA fragments were cloned using the pCR8/GW/

TOPO TA entry vector (Invitrogen) and recombined into the plant transfor-

mation vector pB2GW7 (Karimi et al., 2002). Agrobacterium tumefaciens strain

GV3101 (Koncz and Schell, 1986) containing the pB2GW7 vectors was applied

to Arabidopsis ft-1 mutant flowers using the protocol described by Martinez-

Trujillo et al. (2004). Seeds were sown directly onto soil for selection for

transformants using the Basta herbicide. Putative transformants were con-

firmed by either genomic DNA (gDNA) PCR or qRT-PCR analysis. A

minimum of 10 transformants were characterized for each transgene.

Transformation of Medicago R108 with Agrobacteriumand Regeneration via Somatic Embryogenesis

Four rounds of transformation were conducted with 35S::FTa1 or 35S::GUS

in pB2GW7 (Karimi et al., 2002). Plasmids were transferred into Agrobacterium

GV3101 or EHA105 (Hellens et al., 2000). Explants from 3- to 5-week-old R108-1

(c3) plants growing in vitro in LDs without vernalization were cocultivated

with Agrobacterium containing the constructs and regenerated via somatic

embryogenesis according to the Medicago handbook (Cosson et al., 2006).

Selection for transformant plants was with Basta (3 mg L21; Kiwicare) or DL-

phosphinothricin (2 or 3 mg L21; Sigma). Explants without Agrobacterium

infiltration went through somatic embryogenesis on selective or nonselective

medium as negative control or regeneration control, respectively. Regenerated

T0 transformed plantlets were further cultivated in soil.

RNA Extraction, cDNA Synthesis, and qRT-PCR

RNAwas extracted from approximately 100 mg of plant tissue using either

the RNeasy Plant Mini Kit (Qiagen) followed by TURBO DNase on-column

treatment (TURBO DNA-free Kit; Applied Biosystems) or using Plant RNA

purification reagent (Invitrogen) and subsequent DNase treatment (Invitro-

gen) as per the manufacturer’s instructions. Total RNA (0.5–1 mg) was

transcribed into cDNAwith SuperScript III reverse transcriptase (Invitrogen)

using the (dT)17 primer (Frohman et al., 1988) or with Transcriptor Reverse

Transcriptase (Roche) and random hexamers, according to the manufacturer’s

guidelines. To determine relative gene expression levels, qRT-PCR was

performed in triplicate using either the Roche LightCycler 480 instrument

or the Applied Biosystems 7900HT Sequence Detection System. For the Roche

LC480, qRT-PCR was performed in 10-mL reaction volumes using the Light-

Cycler 480 SYBR Green I Master (Roche). cDNAwas diluted 30-fold, and 3 mL

was used in each reaction. Final primer concentrations were 0.5 mM, and all

primer combinations showed efficiencies greater than 1.8 using an annealing

temperature of 58�C. For qRT-PCR performed using the AB 7900HT Sequence

Detection System, 2 mL of a 20-fold-diluted solution of cDNA sample was

used in a total reaction volume of 10 mL of 13 SYBR Green PCR Master Mix

(Applied Biosystems) with final primer concentrations of 0.5 mM. Relative gene

expression levels were calculated using the 2(2delta delta C(T)) method

(Livak and Schmittgen, 2001) with modifications (Bookout and Mangelsdorf,

2003). Primers used for qRT-PCR experiments can be found in Supplemental

Table S1.

Reverse Genetic Screening for Tnt1 Insertions

Using a reverse genetic PCR approach, Tnt1 insertions were identified in

genes of interest using Tnt1- and gene-specific primers, with superpooled

DNA extracted from over 5,000 Tnt1 mutant lines (Tadege et al., 2008).

Following amplification from superpooled DNA, nested gene-specific primers

and additional rounds of PCR were used to identify the exact line carrying the

Tnt1 insertion in the gene of interest. PCR products were purified and se-

quenced. Gene-specific primer sequences can be found in Supplemental Table S1.

Sequence data from this article can be found at the GenBank/EMBL/DDBJ

data libraries under the following accession numbers:MtFTa1 gDNA, AC123593

(Mt3.5, Medtr7g084970.1, chr7:25432978.0.25435370); MtFTa2 gDNA, AC123593

(Medtr7g085020.1);MtFTc gDNA, AC123593 (Mt3.5, Medtr7g085040.1, chr7:

25463178.0.25466505); MtFTb1 gDNA, AC167329 (Mt3.5, Medtr7g006630.1,

chr7:979293.0.981445); MtFTb2 gDNA, AC167329 (Mt3.5, Medtr7g006690.1,

chr7:1019320.0.1022742); MtFTa1 cDNA, HQ721813; MtFTa2 cDNA, HQ721814;

MtFTb1 cDNA, HQ721815; MtFTb2 cDNA, HQ721816; and MtFTc cDNA,

HQ721817.

Supplemental Data

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

Supplemental Figure S1. Genomic structures of the Medicago FT genes,

and alignment of Arabidopsis and Medicago FT protein sequences.

Supplemental Figure S2. Medicago FT expression in leaves of different

developmental stages.

Supplemental Figure S3. Independent replication of the diurnal expres-

sion profile of MtFT genes in LD and SD conditions.

Supplemental Figure S4. Photoperiodic regulation of MtFT gene expres-

sion.

Supplemental Figure S5. Flowering time and transgene expression levels

in 10 Arabidopsis ft-1 lines expressing MtFTb1 or MtFTb2.

Supplemental Figure S6. Phenotypes of ft-1 Arabidopsis plants over-

expressing MtFTc.

Laurie et al.




Supplemental Figure S7. Characterization of the fta1 mutant alleles.

Supplemental Figure S8. Altered expression of Medicago FTs in the fta1

mutant.

Supplemental Figure S9. Characterization of the ftc mutant alleles.

Supplemental Table S1. A list of primers used in this study.

ACKNOWLEDGMENTS

We thank Jane Campbell and Robyn Lough for technical assistance and

Pascal Ratet for very helpful Medicago transformation advice and providing

R108-1 (c3) seed.

Received May 18, 2011; accepted June 12, 2011; published June 17, 2011.

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the medicago flowering locus t homolog, …...the medicago flowering locus t homolog, mtfta1,...

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