FT regulation of Medicago flowering Corresponding Author: NEW … · production of a mobile floral...

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Running title:

FT regulation of Medicago flowering

Corresponding Author:

Richard Macknight

Department of Biochemistry

University of Otago

PO Box 56

Dunedin

NEW ZEALAND

Phone +64 3 479 5149

Email [email protected]

Journal Research Area

Development

Plant Physiology Preview. Published on June 17, 2011, as DOI:10.1104/pp.111.180182

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The Medicago truncatula FLOWERING LOCUS T homologue, MtFTa1, is a key

regulator of flowering time

Rebecca E. Laurie 1, Payal Diwadkar 1, Mauren Jaudal 1, Lulu Zhang 2, Valérie Hecht 3,

Jiangqi Wen 4, Million Tadege 4, a, Kirankumar S. Mysore 4, Joanna Putterill 2, James L.

Weller 3 and Richard C. Macknight 1*

1Department of Biochemistry, University of Otago, Dunedin, New Zealand. 2 School of Biological Science, University of Auckland, Auckland, New Zealand. 3 School of Plant Science, University of Tasmania, Hobart, Tasmania, Australia. 4 Plant Biology, Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

Footnotes:

Financial Source

This work was supported by a grant from New Zealand Foundation for Research

Science and Technology (contract C10X0704) and AGMARDT Post doctoral

Fellowship (to R. L.).

a Present Address;

Million Tadege

Oklahoma State University�

Department of Plant and Soil Sciences

Stillwater, Oklahoma, USA

* Corresponding Author;

[email protected]



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ABSTRACT

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral

signal, florigen. In this study, we characterized five FT-like genes from the model

legume, Medicago truncatula (Medicago). The different FT genes showed distinct

patterns of expression and responses to environmental cues. Three of the FT genes

(MtFTa1, MtFTb1 and MtFTc) were able to complement the Arabidopsis thaliana ft-1

mutant suggesting they are capable of functioning as florigen. MtFTa1 is the only one of

the FT genes that is upregulated by both LDs and vernalization, conditions that promote

Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene

flowered very rapidly. The key role MtFTa1 plays in regulating flowering was

demonstrated by the identification of fta1 mutants that flowered significantly later in all

conditions examined. fta1 mutants do not respond to vernalization, but are still responsive

to LDs, indicating that the induction of flowering by prolonged cold acts solely through

MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly

MtFTb1 which is only expressed in leaves under LD conditions and therefore might

contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is

unclear, as the ftc mutants did not have any obvious flowering time or other phenotypes.

Overall, this work reveals the diversity of the regulation and function of the Medicago FT

family.



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INTRODUCTION

To precisely control the timing of flowering, plants have evolved mechanisms to

integrate seasonally predictable environmental cues (such as changes in photoperiod and

prolonged periods of cold temperatures) and developmental cues (such as maturity)

(Amasino, 2010). To allow this diversity of floral cues to influence when flowering

occurs in Arabidopsis thaliana (Arabidopsis), multiple pathways converge on a small

number of genes, the floral integrator genes, including the floral promoters FLOWERING

LOCUS T (FT) and TWIN SISTER OF FT (TSF) (Amasino, 2010). FT and TSF are

members of a family of proteins that contain a phosphatidylethanolamine-binding protein

(PEBP) domain (Kardailsky et al., 1999; Kobayashi et al., 1999). In addition to the FT-

like proteins, the plant PEBP family consists of two other phylogenetically distinct

groups of proteins; the TERMINAL FLOWER 1 (TFL1)-like proteins, and the MOTHER

OF FT AND TFL (MFT)-like proteins (Bradley et al., 1997; Mimida et al., 2001; Yoo et

al., 2004; Yamaguchi et al., 2005; Yoo et al., 2010). FT and TSF, act redundantly to

promote flowering under long day photoperiods (Michaels et al., 2005; Yamaguchi et al.,

2005; Jang et al., 2009). The B-box zinc finger transcription factor CONSTANS (CO)

protein induces the expression of TF and TSF in the vascular tissues under LD inductive

conditions (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000; An et al.,

2004). The LD-specific production of CO protein is achieved through the coincidence of

the circadian expression of CO mRNA and the stabilization of the CO protein in the light

(Suárez-López et al., 2001; Valverde et al., 2004). FT and TSF proteins, produced in the

phloem (Takada and Goto, 2003; Yamaguchi et al., 2005), are transported to the apex

where they are able to dimerize with the bZIP transcription factor, FD, to activate the

expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)

(Michaels et al., 2005; Yoo et al., 2005) and the floral meristem identity genes

APETALA1 (AP1) and LEAFY (LFY) (Abe et al., 2005; Wigge et al., 2005). Thus,

FT/TSF (although movement of TSF is yet to be demonstrated) constitute the long sought

after mobile floral signal molecule, florigen (Corbesier et al., 2007; Jaeger and Wigge,

2007; Mathieu et al., 2007; Notaguchi et al., 2008).



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It is likely that FT-like genes play a universal role in regulating the flowering

time. Evidence for this comes from experiments showing that overexpression of FT

homologues cause very early flowering in eudicot plants, such as tomato (Solanum

lycopersicum; (Lifschitz et al., 2006), hybrid aspen trees (Populus tremula crossed with

P. tremuloides or P. alba; Böhlenius et al., 2006; Hsu et al., 2006), apple (Malus x

domestica; Tränkner et al., 2010) and morning glory (Ipomoea nil; Hayama et al., 2007)

and monocot plants, such as rice (Oryza sativa; Kojima et al., 2002; Izawa et al., 2002)

and wheat (Triticum aestivum; Yan et al., 2006). Unlike in Arabidopsis where there are

just two FT-like genes that act redundantly to regulate flowering, other species contain

multiple FT-like genes and many do not appear to play a role in flowering (Cháb et al.,

2008; Komiya et al., 2008; Hagiwara et al., 2009; Blackman et al., 2010; Kotoda et al.,

2010; Kong et al., 2010; Pin et al., 2010). For example, there are 13 FT-like genes in rice

(Chardon and Damerval, 2005), although only two, RFT1 and Hd3a, have been shown to

regulate flowering time (Kojima et al., 2002; Komiya et al., 2008; Komiya et al., 2009).

In addition to their roles in regulating flowering time, FT-like genes can also affect other

aspects of plant development. In tomato (Lycopersicon esculentum), a balance between

levels of the FT orthologue SINGLE FLOWER TRUSS (SFT) and TFL1 orthologue

SELF-PRUNING (SP) determines a range of phenotypes, including stem girth and leaf

development (Shalit et al., 2009). In hybrid aspen trees (Populus temula x tremuloides) an

FT-like gene regulates both the LD induction of flowering and also the SD induced

growth cessation and bud set that occurs in the autumn (Böhlenius et al., 2006). Thus, the

FT-like proteins appear to play a wider role in controlling plant development than just

regulating flowering time.

Crop and forage legumes are second only to grasses in worldwide economic

importance, with flowering time having a significant impact on production yields in

many systems. It is therefore important to understand the molecular-genetics basis of how

flower time is regulated in legumes. Garden pea (Pisum sativum), a long day legume, has

been used in early work on the genetic control of mobile floral signals (Weller et al.,

1997). More recent work in pea has shown that LATE BLOOMER 1 and DIE

NEUTRALIS, orthologues of the circadian clock-associated Arabidopsis genes

GIGANTEA and EARLY FLOWERING 4, respectively, are required for the LD-specific



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production of a mobile floral signal(s) that probably comprise at least two FT-like

proteins (Hecht et al., 2007;Liew et al., 2009; Hecht et al., 2011). Five FT-like genes have

been identified in pea and these genes have different patterns of expression (Hecht et al.,

2011). Hecht et al. (2011) have shown that the pea FTa1 gene corresponds to the GIGAS

locus, which encodes a mobile floral signal that is essential for flowering under long days

and promotes flowering under short days, but is not required for photoperiodic response.

They also provide evidence for a second mobile floral signal that correlates with the

expression of another FT-like gene, FTb2. A recent paper also implicates two FT-like

genes in the regulation of flowering of the short-day legume, soybean (Glycine max)

(Kong et al., 2010).

Here, we characterize five FT genes from Medicago and show that their

expression differs in tissue-specificity, diurnal rhythmicity and response to photoperiod

and vernalization. We find that one of the FT orthologues, FTa1 plays a major role in

regulating flowering time. This gene is essential for the induction of flowering in

response to vernalization but not for the photoperiodic induction of flowering. This work

provides a foundation for understanding the roles of the Medicago FT-family members in

plant development and highlights differences between the behavior FT-family members

in Medicago and pea.



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RESULTS

Medicago has five FT-like genes

Our database searches for putative Medicago truncatula flowering time genes revealed

five FT-like genes, MtFTLa-e (Hecht et al., 2005; Liew et al., 2009; Yeoh et al., 2011).

Phylogenetic analysis of FT/TFL proteins from other plants showed that all five

Medicago FTs belonged to the FT clade and that, in legumes, this clade consists of three

groups of FT proteins, which we have called groups FTa, FTb and FTc (Figure 1A). The

Medicago FTs have been renamed accordingly (FTLa as FTa1, FTLb as FTa2, FTLd as

FTb1, FTLe as FTb2 and FTLc as FTc) (Hecht et al., 2011). The Medicago FT genes

MtFTa1, MtFTa2 and MtFTc are located next to each other within a single BAC clone

(Genbank accession: AC123593) mapping to chromosome 7 and MtFTb1 and MtFTb2

are found in tandem on another chromosome 7 BAC clone (AC127169). The FT-like

genes have a similar genomic structure to the FT genes from other species, with four

exons and three introns (Supplemental Figure S1A) and RT-PCR confirmed that all five

FT-like genes are expressed. An alignment of the predicted protein sequences of the

Medicago FT family is shown in Supplemental Figure S1B.

To examine how widespread the three FT groups are, we constructed a

phylogenetic tree of representative FT protein sequences from the Rosids clade of eudicot

plants. Rosids consist of 17 plant orders, including the Fabales to which the legume

family (Fabaceace) belongs (AGP III, 2009). The FTa, FTb and FTc groups were only

found in the legumes and not in any of the other Rosid orders (Figure 1A). While many

of plants belonging to these orders contain multiple FTs, these are more related to each

other, than the FT within a different order, suggesting that they arose from gene

duplications that occurred after ancestor of the order evolved. The exception being

Rosales, where RcFT (Rosa chinesis; china rose) and FvFT (Fragaria vesca; woodland

strawberry) do not group with the other Rosaceace family FTs (Figure 1A). The Fabales

consist of three other plant families, however, FT sequences are only available from the

Fabaceace family. It is therefore not known if the FTa, FTb and FTc groups are present in

the other Fabale families or if they are legume specific.

The difference between Arabidopsis FT, which promotes flowering, and TFL,



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which represses flowering, has been has been attributed to sequences within an external

loop of protein sequences that contains 14 amino acids (known as segment B) that are

highly conserved within FTs but not in TFLs. A glutamine at position 140 within this

region, as well as an adjacent tyrosine residue (Tyr85) are critical for FT function and are

predicted to form one wall of the ligand-binding pocket (Hanzawa et al., 2005; Ahn et

al., 2006). Analysis of these regions from the legume FT groups and other FTs, revealed

that while these sequences are highly conserved in the other Rosid FTs, the legume FTs

differ from the consensus at 3-6 positions, with the FTc group proteins possessing a

histidine rather than a glutamine at the position equivalent to 140 (Figure 1B). However,

it is not easy to predict from the segment B sequences which of the Medicago FTs might

function to promote flowering.

FT family members show different patterns of expression

To gain insight into the potential roles of the different Medicago FT family members, we

analysed their transcription profiles. Total RNA was isolated from a range of tissues

(Figure 2A and 2B) obtained from plants grown under inductive conditions (seedlings

were vernalized for 14 days at 4 °C and then grown under LDs (16h light: 8h dark); and

qRT-PCR was performed (Figure 2C). All five FT genes were expressed. The highest

level of MtFTa1 transcript was detected in the first leaf (monofoliate leaf) of seedlings,

with strong expression also observed in expanded trifoliate leaves (but not in the

immature ‘folded’ trifoliate leaves), stem and flowers. MtFTa1 was also detected at low

levels in apical bud tissues (this includes the lateral meristem and emerging leaves),

flower buds and developing seedpods, roots and cotyledons (Figure 2C). MtFTa2 was

detected in all tissues examined, except cotyledons. Both MtFTb1 and MtFTb2 were

strongly expressed in the expanded trifoliate leaf and monofoliate leaf but like MtFTa1,

not expressed in the immature trifoliate leaf. MtFTb1 and MtFTb2 were expressed at

moderate and low levels in cotyledons, respectively. MtFTb1 was not detected in the

other tissues examined, while MtFTb2 was expressed at very low levels in these tissues.

MtFTc expression was detected, albeit at low levels, in apical buds, and just detectable

levels of MtFTc transcript were also found in stem, buds and flowers, but were not

detected in leaf or other tissues from vernalized LD plants (Figure 2C). However, MtFTc



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was detected in both leaves and apical buds in SD grown plants sampled at ZT12 (the

time of maximal MtFTc expression, as shown in Figure 5A iv) (Supplemental Figure

S2). Thus, although the five FT genes have distinct patterns of expression they are all

expressed in the leaves, the major site of FT/florigen production in other species (Turck

et al., 2008).

We then examined if the expression of the FT genes changes over a

developmental time course, using vernalized whole plants grown in a LD photoperiod.

MtFTa1 remained relatively constant throughout development (Figure 3A), whereas

MtFTa2 and MtFTb1 were expressed at the highest levels in 5 day old plants (Figure 3B

and C). MtFTb2 transcript levels increased prior to flowering and then decreased about

the time when flowers first appeared (Figure 3D). Given MtFTc is expressed in apical

buds; we examined its expression in this tissue over time (Figure 3E). MtFTc expression

increased up to day 15, the time when the Medicago floral inflorescence identity gene,

PROLIFERATING INFLORESCENCE MERISTEM (PIM) (Benlloch et al., 2006) was

first expressed (Figure 3F), and then gradually decreased. We also examined expression

of the FT family (excluding FTc) in leaves of different ages. Interestingly, levels of

MtFTa1 tended to increase as the leaf aged, whereas, MtFTb1 expression clearly

decreased with leaf age (Supplemental Figure S2). Overall, our results show that, despite

differences in the developmental profile, all five of the FT genes are expressed prior to

the induction of meristem identify gene PIM and therefore are potential regulators of the

floral transition.

Only MtFTa1 is induced both by vernalization and LD photoperiod, conditions that

promote flowering

Next, we investigated if the expression of any of the Medicago FT family members were

induced by the environmental conditions that induce flowering. Flowering in Medicago is

promoted by both exposure to prolonged periods of cold (vernalization) and LD

photoperiods (Clarkson and Russell, 1975). Under our conditions, vernalized Medicago

truncatula cv. Jester flowered 35 and 56 days earlier than non-vernalized plants, when

grown in LD or SD photoperiods, respectively (Figure 4A). LDs also promoted flowering

with non-vernalized, and vernalized plants flowering 56 and 35 days earlier, respectively,



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when grown in LD photoperiod, compared with plants grown in SD photoperiod (Figure

4A). These differences were apparent when flowering time was measured as either days

to produce the first flower or number of nodes produced at flowering (Figure 4A).

We then examined the effect of vernalization and daylength on the expression of

the FT family members. Comparison of the FT gene expression in LD or SD photoperiod

revealed that while MtFTa1 was expressed in SD conditions, it was expressed at ~2-fold

higher levels in LDs (Figure 4B). In contrast, MtFTa2 showed the opposite response with

moderate expression under LD and ~7-fold higher expression under SD conditions. The

expression of MtFTb1 and MtFTb2 was strongly influenced by photoperiod, with both

being expressed in LDs but undetectable in SDs. MtFTa1 and MtFTa2 were strongly

induced by vernalization, with neither gene being expressed at a detectable level in LD

non-vernalized whole plants at day 15 but expressed at high levels in LD vernalized

plants (Figure 4C) [Note; MtFTa1 is expressed in older non-vernalized plants (Figure

8E)]. In contrast, MtFTb1 and MtFTb2 were expressed in non-vernalized and vernalized

plants (Figure 4B). MtFTc expression was not detected using the RNA isolated from

whole plants in this experiment under any of the conditions, although it was detected in

apical buds harvested from LD and SD grown plants (Figure 5A iv).

A feature of the vernalization response is that plants retain an epigenetic memory

of the cold. To examine if prolonged exposure to the cold directly activates the MtFTa1

or MtFTa2 genes, or if subsequent exposure to inductive photoperiod is needed, we

carried out the following experiment: Medicago seeds were germinated, vernalized at 4

°C in the dark for 14 days and then transferred to inductive conditions (21 °C and LD

photoperiod). MtFTa1 was not expressed in germinated seedlings after 0, 7 or 14 days in

the cold, however, after being transferred into warm LD conditions for 1 day, MtFTa1

expression was just detectable, with expression increasing after 7 and 14 days (Figure

4D). Thus, vernalization per se does not induce MtFTa1 expression, implying that

Medicago retains a ‘memory’ of being exposed to cold. Surprisingly, MtFTa2 responded

quite differently and was upregulated after 14 days at 4 °C while the seedlings were still

in the cold and dark. This upregulation of MtFTa2 expression was maintained after the

plants are returned to the warm (Figure 4E).



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In summary, the four leaf-expressed FT genes are induced by conditions that

promote flowering (MtFTa1 and MtFTa2 by vernalization and MtFTa1, MtFTb1 and

MtFTb2 by LD photoperiod) and therefore may play a role in the induction of flowering;

however, only MtFTa1 is induced by both vernalization and LD photoperiod.

Transfer from a SD to LD photoperiod promotes flowering and leads to the rapidly

upregulation of MtFTa1, MtFTb1 and MtFTb2

To further investigate the affect of photoperiod on the expression of the FT family

members, we compared their expression over a diurnal time course from LD and SD

grown plants (Figure 5A and Supplemental Figure S3 for independently replicated data).

MtFTa1 was expressed at similar levels throughout the LD and SD diurnal time course

and did not appear to cycle in a robust manner (Figure 5A i). However, consistent with

Figure 4B, MtFTa1 was found to be expressed at significantly higher levels in LDs,

compared with SDs (Figure 5A i). MtFTa2 was also detected at all time points in both the

LD and SD diurnal time course, and was the highest expressed FT family member under

SD conditions, with peak expression occurring during the dark (ZT12) (Figure 5A ii).

MtFTb1 expression was not detected at any time points of the SD diurnal time course but

expressed bimodally in LDs, with peak expression at 4 hours after dawn (ZT4) and at

dusk (ZT16) (Figure 5A iii). MtFTb2 showed a similar pattern of expression

(Supplemental Figure S3). Although MtFTc is expressed at very low levels in whole

plants, like MtFTa2, it was also expressed at its highest level in SDs at ZT12 (Figure 5A

iv). We were unable to detect MtFTc expression in the leaves of LD plants, however, give

that maximal MtFTc expression was detected in SDs at ZT12, we examined whether it is

expression in the leaves at this time point. The revealed that MtFTc is expressed, albeit

still at relatively low levels, in both the leaves and apical tissues in SDs (Supplemental

Figure S2). Overall, these results demonstrate that the different Medicago FT family

members have distinct photoperiodic responses.

To determine if any of the FT family members are regulated by photoperiod

within a time frame that is consistent with them having a role in regulating flowering, we

performed an experiment where Medicago plants were grown under a SD photoperiod

and then shifted for varying number of days, into inductive LD conditions. Plants grown



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in SD conditions after vernalization flowered with about nine nodes. When these plants

were shifted into a LD photoperiod when they had one visible node (and many other

nodes already formed within the shoot apex), they flowered with a total of about seven

nodes (Supplemental Figure S4A). Shifting these plants from SD into LDs for just one

day was not sufficient to promote flowering. However, flowering was promoted when the

plants were shifted into LDs for 3 or more days (Supplemental Figure S4A). This

indicates that three LDs were sufficient to induce flowering in plants of this age. Next,

we examined if the expression of different FT family members responded to changes in

photoperiod on a similar time scale. RNA obtained from M. truncatula cv. R108 leaves

and apical buds collected at ZT4 after plants were grown in SD, then transferred to LDs

for 1, 2 and 3 days, then back to SD for 1, 2, and 3 days (a similar experiment using RNA

obtained from M. truncatula cv. Jester whole plants collected at ZT2 is shown in

Supplemental Figure S4). MtFTa1 was upregulated in both the leaves and apical buds

after just one long day and its expression increased after a further 2 and 3 days in LDs

(Figure 5B i). MtFTa1 returned to pre-shift SD levels after just one day after being

transferred back into SDs (Figure 5B). Interestingly, MtFTa2 showed the opposite pattern

of expression to MtFTa1 and was expressed at lower levels in both leaves and in apical

buds when grown in LD photoperiod compared with the once the plants were shifted

back into SDs (Figure 5B). MtFTb1 and MtFTb2 also responded rapidly to changes in

photoperiod with one LD being sufficient for their up-regulation and further increases

seen after 2 and 3 LDs (Figure 5B). MtFTb1 and MtFTb2 were not detectable when

returned to SDs (Figure 5B). Thus, MtFTa1, MtFTb1 and MtFTb2 are up-regulated

rapidly by LDs and therefore could be responsible for the LD induction of flowering. The

different behavior of MtFTa1 and MtFTa2 in the shift experiments suggests that the two

genes are regulated by different mechanisms, and this is consistent with their different

diurnal pattern of expression and vernalization response.

MtFTa1, MtFTb1 and MtFTc encode FT proteins that can complement the

Arabidopsis ft-1 mutant

To assess the potential of MtFT genes to regulate flowering time, the five Medicago

genes were over expressed in Arabidopsis using the CaMV 35S promoter. The 35S:MtFT



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constructs were introduced into the late-flowering Arabidopsis ft-1 mutant plants (in the

Ler background). At least ten independent homozygous T3 lines were produced and

representative plants grown under LD conditions are shown in Figure 6A. 35S:MtFTa1

complemented the ft-1 mutation and caused plants to flower even earlier than wild-type

Ler plants, with 35S:MtFTa1 plants flowering with 5.0 ± 0.2 leaves compared with 9.1 ±

0.9 leaves for Ler. In contrast, plants overexpressing MtFTa2 flowered with 22.4 ± 0.6

leaves, which was similar to ft-1 (21.4 ± 2.0). MtFTb1 was able to partially complement

ft-1 with high expressing lines (such as the line shown in Figure 6) flowering with similar

number of leaves as Ler, whereas other lines showed intermediate flowering with up to

15 leaves (Supplemental Figure S5A). In contrast, 35S:MtFTb2 lines were unable to

complement ft-1, even when expressed at high levels (Figure 6 and Supplemental Figure

S4B). 35S:MtFTc plants flowered very early, with some lines flowering without

producing any rosette leaves and only 2 cauline leaves. The 35S:MtFTc plants showed

other abnormalities, such as curly leaves (Supplemental Figure S6) that have been found

in 35S:AtFT Arabidopsis plants (Teper-Bamnolker and Samach, 2005). In summary,

MtFTa1 and MtFTc can fully complement ft-1 and result in very early flowering when

driven by the 35S promoter, whereas MtFTb1 partially complements ft-1.

Overexpression of MtFTa1 strongly accelerates flowering in transgenic Medicago

plants

M. truncatula cv. R108 was transformed with the same 35S:MtFTa1 construct that was

used to complement the Arabidopsis ft-1 mutant. As controls, we transformed plants with

a 35S:GUS construct and also regenerated Medicago plants from leaf explants that had

not been infected with Agrobacterium. The flowering time of T0 plants (plants

regenerated directly from explants) was measured. Nine independently generated

35S:MtFTa1 plants were obtained and all flowered significantly earlier than the 35S:GUS

and regenerated plant controls (Figure 7A and C). In non-vernalized plants MtFTa1 was

expressed at ~30 to ~3400-fold higher levels in the three 35S:MtFTa1 lines examined,

compared with the 35S:GUS or regenerated control plants (Figure 7B). We then

measured the flowering time of T1 progeny from two of the 35S:MtFTa1 lines grown in

LD photoperiod without vernalization (Figure 7 D and E). All 12 progeny from the first



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line, M1-4, flowered significantly earlier (average 28.6 ± 0.9 days) than R108 (average

54.1 ± 1.1 days). The second line segregated early flowering plants that flowered after

just 24 ± 0 days with only two nodes and a late flowering plant that flowered after 57

days with 11 nodes. Thus, MtFTa1 can induce flowering when overexpressed in

Medicago, even in the absence of vernalization, indicating FT expression is a limiting

factor in promoting the floral transition. The 35S:MtFTa1 plants also exhibited increased

internode elongation and less branching compared to the 35S:GUS and regenerated

control plants, resulting in a more erect phenotype, indicating that in addition to

promoting flowering, MtFTa1 influences other aspects of plant development.

Medicago fta1 mutants are late flowering

The fact that MtFTa1 is expressed in the leaves, is upregulated by conditions that

promote flowering (vernalization and LDs), and causes early flowering when

overexpressed in Arabidopsis and Medicago, strongly implicates MtFTa1 as a key

regulator of flowering. To provide more direct evidence for the role of MtFTa1 in

flowering, we set out to identify plants carrying mutations in this gene. Using M.

truncatula cv. R108 Tnt1 retrotransposon tagged insertion mutants (Tadege et al., 2008),

a reverse genetic approach was employed to screen for Tnt1 insertions within MtFTa1

gene using PCR. Two different lines were found to have Tnt1 sequences within the

MtFTa1 gene. Line NF3307 contained an insertion within exon 1 (fta1-1) and Tnt1 was

inserted within intron 1 in line NF2519 (fta1-2) (Figure 8A). Both lines segregated plants

with a late flowering phenotype (fta1-1 is shown Figure 8B and fta1-2 is shown in

Supplemental Figure S7). No RT-PCR products were detected using primers across the

insertion sites indicating that the Tnt1 sequences prevent a correctly spliced mRNA from

being produced (Figure 8C). Next, we investigated if late flowering correlated with

homozygous Tnt1 insertions. In both lines, ~25% of the plants were late flowering and all

were homozygous for the Tnt1 insertion within the MtFTa1 gene (Supplemental Figure

S7). Together, these results indicate that disrupting the MtFTa1 gene results in a late

flowering phenotype. Loss of FTa1 also affected the growth habit (Figure 8B). In

comparison to WT, the fta1 mutant showed dramatically reduced elongation of main stem



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internodes, more profuse and vigorous lateral branching at lower nodes, and a more

prostrate habit overall.

To investigate the environmental conditions in which MtFTa1 functions to

promote flowering, wild type R108 (from NF3307) and the fta1-1 mutant were either

vernalized for two weeks at 4 °C or not vernalized and grown in an LD or SD

photoperiod. The vernalized fta1-1 mutant flowered later than wild type when grown in

both LD and SD photoperiods, consistent with the expression data showing that while

MtFTa1 is upregulated by LD photoperiod, it is also expressed in SD conditions (Figure

4C and 5). However, the fta1-1 mutant still flowered significantly earlier when grown in

LD compared with SD photoperiods (92 days c.f 141 days Figure 8D). Similarly, the

non-vernalized fta1-1 mutant also flowered earlier in LD compared with SD conditions

(94 days c.f 144 days) (Figure 8D). Therefore, while the MtFTa1 gene plays a major role

in the promotion of flowering under a LD photoperiod, an MtFTa1-independent pathway

must also exist to allow Medicago to flower earlier in LD conditions.

In both LD and SD conditions, the vernalized fta1-1 mutant flowered at the same

time as the non-vernalized fta1-1 mutant (Figure 8D). Thus, the fta1-1 mutant is

completely unresponsive to vernalization indicating that MtFTa1 is the major target of

the vernalization pathway. Given that we did not detect MtFTa1 expression in non-

vernalized 15 day old plants, it was surprising that the fta1-1 mutant flowered later than

wild-type plants without vernalization. We therefore examined MtFTa1 expression in

older non-vernalized plants and found that MtFTa1 was indeed expressed in plants prior

to flowering (Figure 8E). Thus the expression of MtFTa1 in non-vernalized plants later in

development is sufficient to promote flowering.

Altered expression of FT family members in fta1

To investigate if MtFTa1 affects the expression of any of the other FT genes, we first

examined the expression of MtFTa2, MtFTb1 and MtFTb2 in fta1-1 mutant plants grown

in SD and shifted in to LD photoperiod. This experiment was performed at the same time

as we conducted the shift experiment with wild-type plants shown in Figure 5B. The fact

that MtFTa1 is expression at higher levels in LD than SD photoperiod whereas MtFTa2

shows the opposite expression pattern (Figure 5), raised the possibility that MtFTa1



16

might repress MtFTa2 expression. However, MtFTa2 was expressed in both the leaves

and nodes in fta1-1 mutant plants and at similar levels to that in wild-type plants (Figure

9A). Therefore, the reduced levels of MtFTa1 in SDs is not responsible for the rapid

upregulation of MtFTa2 that occurs when plants are shifted back to SDs (Figure 9B).

Similarly, it was possible that the upregulation of MtFTa1 in LDs might contribute to the

expression of MtFTb1 and MtFTb2 only under LDs. However, both MtFTb1 and MtFTb2

were upregulated upon transfer from SD to LD conditions in the fta1-1 mutant, indicating

FTa1 is not required for their expression in LDs (Figure 9C).

To further investigate the regulation of the FT genes, we examined their

expression in wild-type and fta1-1 mutant in leaves and apical bud tissue at different

stages of development. RNA was isolated from plants with two nodes (~10 days), five

nodes (~20 days) and plants that had flowered (~60 days for wild type plants and ~80

days for the late flowering fta1-1 mutant). As expected, MtFTa1 was expressed in both

leaves and apical buds at these time points, but not detected in the fta1-1 mutant

(Supplemental Figure S8A). Prior to flowering, MtFTa2 was comparable between wild

type and fta1-1 in both leaves and apical buds (P=0.45 and P=0.57, respectively) (Figure

9A). However, after flowering MtFTa2 was expressed at significantly higher levels

(P<0.01) in the fta1-1 mutant compared to wild type (~10-fold and ~8-fold higher in

leaves and apical buds, respectively; Supplemental Figure S8B). MtFTb1 was expressed

at slightly higher levels in the leaves of the fta1-1 mutant compared with wild type,

although this difference was not statistically significant (P=0.063), However, after

flowering, MtFTb1 was expressed at significantly high levels in fta1 compared to wild-

type (P=0.014) (Supplemental Figure S8C). In contrast, there were no significant

differences in MtFTb2 between fta1-1 mutant and wild type (P=0.677) (Figure 9C). The

increased expression of MtFTa2 and MtFTb2 in the fta1-1 mutant might be due to the

significant difference in the age of the fta1-1 and wild-type plants when they flowered,

however, it might also be due to MtFTa1, directly or indirectly, suppressing MtFTa2 and

MtFTb1 expression. Additional experiments will be required to establish why MtFTa2

and MtFTb1 are expressed at higher levels in the fta1-1 mutant. However, the effect of

the fta1-1 mutant on MtFTc expression was more dramatic. Prior to flowering, at the 2

and 5 node stages, MtFTc was only expressed in the apical buds of wild-type plants and



17

could not be detected in the fta1-1 mutant (Figure 9D). However, MtFTc expression was

observed once fta1-1 plants had flowered,(Figure 9D). The fact that in LDs, MtFTc is

only expressed in the apical buds and only once the fta1-1 plants flowered, suggests that

MtFTc might be expressed in a similar manner to the floral meristem identity genes. We

examined the expression of MtPIM and a Medicago homologue of FRUITFULL, FULc

(Hecht et al., 2005). Both these genes were not expressed in wild type plants at the 2 node

stage (~10 days after germination), whereas MtFTc was detected at this stage (Figure

9D). Thus, MtFTc is expressed prior to the induction of the floral meristem identity

genes.

ftc mutants have no obvious phenotype

MtFTc is expressed in apical bud tissue prior to the expression of the floral meristem

identity genes and its expression is delayed in the fta1-1 mutant, suggesting that it might

be involved in the floral transition. To investigate the role of MtFTc, we screened the

Tnt1 lines for inserts within the MtFTc gene using the same approach we used to identify

the fta1 mutants. Three independent Tnt1 insertions were identified in MtFTc within the

5’ UTR (NF6335; ftc-1), exon 1 (NF4345; ftc-2) and intron 1 (NF4913; ftc-3) (Figure

10A). The Tnt1 insertions all segregated ~3:1 and homozygous lines for each of the lines

were identified (Supplemental Figure S9). Quantitative RT-PCR using primers to amplify

across MtFTc exons 1 and 2 revealed that MtFTc is expressed at lower levels in ftc-1

homozygous plants, and that in ftc-3 homozygous plants, a low level of correctly-spliced

transcript persists despite the large Tnt1 insert, In contrast, MtFTc was not detectable in

ftc-2 mutant plants, indicating that only ftc-2 is likely to be a null mutant. Homozygous

plants for each of the three Tnt1 insertions developed normally and were phenotypically

indistinguishable from wild type, with normal flowers forming at approximately the same

time (Figure 10B and Supplemental Figure S9). Thus, MtFTc does not appear to play a

major role in regulating Medicago flowering in LDs, consistent with its confined

expression pattern in apical buds. However, given MtFTc is also expressed in the leaves

in SD grown plants (Supplemental Figure S2), we are currently investigating if MtFTc

influences flowering in SDs.

DISCUSSION



18

FT-like proteins appear to play a universal role in regulating flowering time (Turck et al.,

2008). Here, we have characterized five Medicago FT-family members and show that the

different FTs have distinct patterns of expression and responses to environmental cues.

We demonstrate that only one of the FT genes, MtFTa1, is regulated by both

vernalization and a LD photoperiod and this gene plays a major role in the regulation of

Medicago flowering.

Unlike other Rosids, the legumes have three distinct groups of FT proteins, FTa, b

and c (Hecht et al., 2011; Figure 1A). MtFTa1 encodes an FTa group protein and recent

work by the Weller group has shown that the pea FTa group gene, PsFTa1, also plays a

key role in regulating flowering time. PsFTa1 corresponds to the GIGAS locus and is

essential for flowering under LD conditions and promotes flowering under SD conditions

(Hecht et al., 2011). Like Medicago, pea has a least five FT-like genes and these genes

shown distinct patterns of expression. The pea FTb group gene, PsFTb1, is also

implicated in the LD induction of flowering (Hecht et al., 2011). Ten FT genes have been

found in soybean, a short day legume (Kong et al., 2010; Hecht et al., 2011). Two of

these soybean FT genes (GmFT2a and GmFT5a) have recently been implicated in

controlling flowering (Kong et al., 2010). GmFT2a encodes an FTa group protein and

GmFT5a encodes an FTc group protein, named GmFTa3 and GmFTc1, respectively, by

Hecht et al., 2011 (Figure 1A). It therefore appears that flowering in legumes involves

multiple FT genes that belong to different FT groups.

Overexpression of the different Medicago and pea FT-family members in

Arabidopsis ft1-1 mutant revealed that not all the FT-like genes are functional, and even

those that were able to complement ft-1 varied in the degree which they promoted

flowering. Therefore, sequence variation within the FT-like family members affects their

ability to promote flowering. Conserved sequences within the FT proteins are required

for the promotion of flowering, The Tyr85/His88 and Gln140/Asp144 residues, as well as

sequences within external loop of the PEBP proteins, known as segment B, determine

whether the FT-like and TFL1-like members promote or repress flowering (Hanzawa et

al., 2005; Ahn et al., 2006). While the all legume FT-like proteins have the conserved

Tyr85, the legume FTc group proteins, which strongly promote flowering, have a His



19

rather than Gln140. Segment B, which is conserved in the FT-like proteins from other

plants, is more divergent in the legume FTs. Amino acid changes within this region can

have a profound affect the activity of the FT-like proteins. In sugar beet, just three amino

acid changes within this region (Tyr138/Asn134, Gly141/Gln137 and Trp142/Gln138)

determine the ability of BvFT2 and BvFT1 to promotes and repress flowering,

respectively (Pin et al., 2010). These three amino acids are conserved within strongly

activating MtFTa1 and PsFTa1/GIGAS proteins. However, interestingly that two of these

amino acids (equivalent to Tyr138 and Gly141 in BvFT2) vary in the FTc proteins.

Tyr138 is replaced with other polar amino acids (Phe or Ile), whereas in BvFT1 it is

replaced with a non-polar amino acid, Asn. This suggests that while conservative

substitutions at Tyr138 do not abolish FTs ability to promoter flowering, non-

conservative changes might have a more profound affect. In contrast, changes at Gly141,

a non-polar amino acid, are probably less important as the acidic residues, Asp or Glu are

present at the equivalent position in FTc proteins. Thus, it is unlikely that the presence of

the polar amino acid, Gln, at this position in BvFT1, is responsible for the late flowering

properties of BvFT1. All the FTs except BvFT1 have a Trp at the equivalent position to

142 in BvFT2. It is therefore likely that this amino acid change, probably together with

the Try138 to Asn change, are key to BvFT1 repressive function. However, it is not clear

whether the differences in the B segment region between the different FT groups are

responsible for the differing abilities of the FTs to promote flowering. Clearly, other

sequence variation within other regions also affect the ability of the different FTs to

promote Arabidopsis flowering when overexpressed, as MtFTa1/MtFTa2 and MtFTb1/

MtFTb2 have identical B segments, although only MtFTa1 and MtFTb1 cause early

flowering.

Our results indicate that MtFTa1 plays a key role in the promotion of flowering

by vernalization. The expression of MtFTa1 is strongly induced by a vernalization

treatment (Figure 4C) and the fta1-1 mutant appears to be unable to respond to

vernalization (Figure 8D) indicating that MtFTa1 is the major, and possibly only, target

of the vernalization pathway. Although another FT gene, MtFTa2, is also induced by

vernalization, this gene was unable to complement the ft-1 mutation when overexpressed

in Arabidopsis. It therefore appears that MtFTa2 does not encode a protein capable of



20

inducing flowering. The identification and characterization of an MtFTa2 mutant will be

required to establish if it plays any role in Medicago flowering.

A hallmark of the vernalization process is that plants retain an epigenetic memory

of being exposed to the cold. To investigate if upregulation of the MtFTa1 or MtFTa2

genes in response to vernalization actually occurs after the plants are returned to the

warm, we examined their expression in germinated seedlings growing at 4 °C in the dark

and then growing at 21 °C in LDs. MtFTa1 was not expressed in seedlings during the

cold, but was upregulated when the plants were transferred into warm LDs (Figure 4D),

This implies that, like in other plants, Medicago plants retain a memory of the cold.

Interestingly, MtFTa2 expression was upregulated while still in the cold, however, its

expression was maintained when the plants were grown in the warm, suggesting there

might also be a epigenetic basis to its regulation.

Although MtFTa1 is strongly induced by vernalization, MtFTa1 is expressed a

low levels in non-vernalized plants (Figure 8E) and this low level of expression is

sufficient to promote flowering, as the non-vernalized fta1-1 mutant flowered

significantly later than wild type plants (Figure 8D). Thus, although MtFTa1 is the major

target of the vernalization pathway, it also plays an important role in determining the

flowering time of non-vernalized plants. In vernalization-responsive Arabidopsis

accessions, FT expression is repressed prior to vernalization by FLC interacting with the

FT promoter (Searle et al., 2006). Vernalization results in the epigenetic silencing of the

FLC gene, which then allows the FT gene to be upregulated by the photoperiod pathway.

Medicago does not possess an obvious FLC homologue (Hecht et al., 2005) raising the

possibility that the vernalization responsive regulation of MtFTa1 might stem from a

different mechanism from Arabidopsis. This may not be surprising, as it has been

suggested that since flowering plants evolved in a warm environment where vernalization

would not be necessary, the cold induction of flowering might have evolved

independently in different families of flowering plants (Amasino, 2010). In support of

this, a recent study has shown that vernalization of cultivated sugar beet, Beta vulgaris

ssp vulgaris, a eudicot in the family Amaranthaceae, involves a different mechanism

from Arabidopsis, a member of the Brassicaceae (Pin et al., 2010). In sugar beet, rather

than silencing an FLC-like gene, vernalization down-regulates an FT gene, BvFT1, that



21

has evolved the ability to 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

downregulated by vernalization. Therefore, the upregulation of MtFTa1 by vernalization

does not appear to involve another FT. Vernalization in cereals also involves the

downregulation of an FT repressor, in this case a CONSTANS, CONSTANS-like, and

TIMING OF CAB EXPRESSION 1 (CCT)-domain transcription factor VRN2 (Yan et al.,

2004; Hemming et al., 2008). We are currently searching for genes that encode

vernalization responsive repressors of MtFTa1 expression.

The expression of MtFTa1 is induced by a LD photoperiod (Figure 5B), and

consistent with a role in promoting flowering in this condition, the fta1 mutant has

significantly delayed flowering in LDs compared with wild type plants (Figure 8D).

However, the fta1 mutant is still capable of responding to photoperiod, indicating that

another pathway exists. A candidate for this pathway is MtFTb1. Consistent with a

potential role as a florigen, MtFTb1 is expressed in the leaves under LD photoperiods but

is not expressed at detectable levels in SD photoperiods (Figure 5B) and can complement

the Arabidopsis ft-1 mutant when overexpressed at high levels (Figure 6). Unfortunately,

we have not yet identified a Tnt1 insertion within the MtFTb1 gene to examine whether it

is involved in regulating flowering. However, recent work in pea supports the idea that

photoperiodic regulation of flowering in temperate legumes might involve multiple FT

genes (Hecht et al., 2011). Hecht et al. (2011) have shown that PsFTa1 gene corresponds

to the GIGAS gene. The Psfta1/gigas mutant is deficient in a mobile floral signal that is

required for flowering under LD and promotes flowering under SD conditions, however,

the mutant can still respond to photoperiod (Beveridge and Murfet, 1996; Hecht et al.,

2011). Hecht et al. (2011) provided evidence that the photoperiodic induction of

flowering involves a second mobile floral signals that correlates with the expression of

the FTb group gene, PsFTb2, which like the Medicago FTb1 and FTb2 genes, is induced

by LDs. Thus, the photoperiodic induction of flowering of both Medicago and pea might

involve FTa and FTb group FT proteins. Interestingly, MtFTa1 and MtFTb1 are regulated

differently by photoperiod; for example, MtFTa1 lacks a robust diurnal rhythm and is

expressed in SDs, whereas MtFTb1 is regulated diurnally and not expressed in SDs. This

suggests two mechanisms might exist to mediate the LD induction of the FTs.



22

Although the FTa group genes MtFTa1 and PsFTa1/GIGAS play an important

role in the regulation of Medicago and pea flowering, respectively, they do not have

exactly the same function. While flowering of the Medicago fta1 mutant is delayed under

LDs, normal flowers are eventually produced and the meristem identify genes, MtPIM

and MtFULc are expressed in the apical buds at flowering (Figure 9D). In contrast,

Psfta1/gigas mutants generally fail to produce flowers under LDs and this is associated

with a lack of expression of the floral meristem identity genes PIM and SEPALLALA1

and the reduced expression of UNIFOLIATA (Hecht et al., 2011). Interestingly, flowering

of Psfta1/gigas is only delayed under SD photoperiod and normal flowers are produced

(Beveridge and Murfet, 1996; Hecht et al., 2011). The Medicago fta1 mutant also has

delayed flowering under SDs. The role of MtFTa1 in the induction of flowering under

SDs is in contrast to Arabidopsis where FT/TSF play a very minor role in SDs, with the

ft-10 tsf-1 double mutant flowering only slightly later than wild type plants in SDs (Jang

et al., 2009). Instead the SD flowering of Arabidopsis involves the FT/TSF independent

induction of SOC1 by gibberellin (Borner et al., 2000; Jang et al., 2009). Recently, two

FT genes (GmFT2a/FTa3 and GmFT5a/FTc1) have been implicated in the flowering of

the short day legume, soybean (Glycine max). These genes are expressed at low levels in

LDs and upregulated under SDs. Thus, the altered regulation of the FT genes appears to

be a key difference between long- and short-day legumes.

In addition to the LD-induced MtFTa1 and MtFTb1 genes, MtFTc was also able

to complement the Arabidopsis ft-1 mutant. Although more divergent in sequence

compared with the other Medicago FTs, MtFTc caused Arabidopsis to flower very early

when overexpressed. This suggested that MtFTc might play a role in controlling

flowering time. However, the ftc mutants flowered at the same time as wild type and did

not have any obvious phenotypes. Thus, the role of MtFTc is not clear. A possible

explanation for the lack of a flowering-time phenotype in the ftc mutant is that MtFTa1

and MtFTc have overlapping or partially redundant roles and that MtFTa1 activity can

compensate for the lack of MtFTc. However, the fta1/ftc double mutant needed to address

this question will be difficult to obtained, as the two genes are tightly linked (only ~30kb

apart). MtFTc is expressed in the apical buds but not leaves and its expression is

associated with flowering. The fta1 mutant has no detectable MtFTc expression prior to



23

flowering, however, MtFTc is expressed once fta1 plants flower. This may be similar to

the Arabidopsis floral meristem identity gene AP1, which is activated by FT but can also

be activated by other pathways that induce flowering. However, MtFTc expression was

detected prior to the induction of the Medicago orthologue of AP1, MtPIM, and the

meristem identity gene MtFULc. Thus, MtFTc expression appears to be associated with

the floral transition, rather than simply with the formation of flowers. In pea, the PsFTc

gene is also expressed in apical buds, but not in leaves (Hecht et al., 2011). Like

Medicago, the PsFTc gene is upregulated at a similar time to the pea meristem identity

genes and its expression is reduced in the Psfta1/gigas mutant (Hecht et al., 2011). In

contrast to Medicago and pea, the soybean FTc group gene, GmFT5a/FTc1, is thought to

play a role in regulating flowering, as it is upregulated by inductive SD photoperiods and

is expressed in the leaves. Thus, the FTc gene may play a different role in the tropical SD

legume, soybean, compared with the temperate LD legumes, Medicago and pea.

In pea, the expression of the FTa2 gene is abolished in the fta1/gigas mutant,

indicating that FTa1 directly or indirectly upregulates FTa2 expression. However, this is

not the case in Medicago where lower expression of FTa2 is not seen in the fta1 mutant

(Figure 5). If anything, FTa1 might lead to the down-regulate FTa2 as once the plants

flower, FTa2 is expressed at higher levels in the fta1 mutant compared with wild type

plants (Supplemental Figure S8). Other differences also between the pea and Medicago

FTa2 genes. The PsFTa2 can complement Arabidopsis ft-1 (Hecht et al., 2011), whereas

MtFTa2 was unable to complement the Arabidopsis ft-1 mutation, and therefore probably

does not encode a protein capable of affecting flowering. MtFTa2 expression is induced

by vernalization and SDs, environmental conditions that might allow Medicago to adapt

its flowering response to suit particular regions. Therefore more recent mutations within

MtFTa2 might have resulted in the loss of its ability to function as a florigen. It is also

possible that MtFTa2 actually delays flowering in SDs, perhaps by competing with

MtFTa1. Further experiments will be needed to establish if MtFTa2 plays a role in

flowering. Similarly, while MtFTb2, like MtFTb1, is only expressed in LD photoperiods,

MtFTb2 cannot complement Arabidopsis ft-1, while MtFTb1 partially complements ft-1.

Interestingly, in pea both the PsFTb1 and PsFTb2 genes can complement Arabidopsis ft-

1, however, only PsFTb2 is induced by LDs and FTb1 is expressed at very low levels in



24

both LD and SD throughout development (Hecht et al., 2011). Thus, in both Medicago

and pea, probably only one FTb group gene functions in the LD induction of flowering.

Sequences variants within FT homologues from sunflower (Blackman et al., 2010), wheat

(Yan et al., 2006), rice (Hagiwara et al., 2009) and Arabidopsis (Schwartz et al., 2009)

have been shown to be important for flowering time adaptation. Since there is significant

variation in flowering time in different Medicago accessions (Skinner et al., 1999), it is

possible that variation in the activity or expression of the various Medicago FT family

members underlies some of this variation.

In conclusion, this work provides a detailed characterization of the Medicago FT

gene family and demonstrates that MtFTa1 plays a key role in the regulation of

flowering. Since the other FTs family members are also regulated by vernalization and/or

day length, it possible that they also influence flowering. Future work will focus on

understanding the role of the different FT-family members.



25

MATERIALS AND METHODS

Bioinformatics

Medicago truncatula homologues of Arabidopsis FT were identified using tBLASTn

searches against M. truncatula ESTs and BAC sequences. Alignments were performed

were aligned using ClustalW in the Geneious software package. Phylogenetic

relationships among sequences were estimated using the Neighbour-Joining method (NJ)

in MEGA 4.0.2 program (Kumar et al., 2008). The accession numbers for sequences are;

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

AtBFT (NM_125597), AtMFT (NM_101672), Beta vulgaris BvFT1 (HM448910),

BvFT2 (HM448912), Citrus unshiu CiFT (AB027456), CiFT2 (AB301934), CiFT3

(AB301935), CmFT-like2 (ABI94606), Cucurbita maxima CmFT-like1 (ABI94605),

Ficus carica FcFT (BAI60052), Fragaria vesca FvFT (CBY25183), Glycine max

GmFTa1 (AB550124; Glyma16g04840), GmFTa2 (AB550125; Glyma19g28390),

GmFTa3/FT2a (AB550122; Glyma16g26660), GmFTa4 (Glyma16g26690), GmFTb1

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

Glyma18g53680), GmFTb4 (AB550121; Glyma18g53690), GmFTc1/5a (AB550126;

Glyma16g04830), GmFTc2 (Glyma19g28400), Gossypium hirsutum GhFT (HM631972),

Malus x domestica MdFT1 (AB161112), MdFT2 (AB458504), Medicago truncatula

MtFTa1 (HQ721813), MtFTa2 (HQ721814), MtFTb1 (HQ721815), MtFTb2

(HQ721816), MtFTc (HQ721817), MtTFL1 (Medtr7g127250), MtBFT (AC146807_6.1),

MtMFT (Medtr4g155400), Oryza sativa OsHd3a (NM_001063395), Populus nigra

PnFT1 (AB106111), PnFT2 (AB109804), PnFT3/4 (AB110612), Poncirus trifoliate PtFT

(EU400602), Prunus mume PmFT (BAH82787), Pisum sativum PsFTa1 (HQ538822),

PsFTa2 (HQ538823), PsFTb1 (HQ538824), PsFTb2 (HQ538825), PsFTc (HQ538826),

Pyrus pyrifolia PyFT (BAJ11577), Rosa chinensis RcFT (CBY25182), Solanum

lycopersicum LeSP3D (AY186735), and Vitis vinifera VvFT-like (DQ871590).

Plant materials and growth conditions



26

Medicago truncatula var Jester was kindly supplied by Seedmark (Australia) and

accession R108 (Hoffmann et al., 1997) and various Tnt1 insertion lines were obtained

from the Noble Foundation (USA). Medicago and Arabidopsis plants were grown under

the following regimes; 16h light/8h dark for LDs and 8h light/16h dark for SDs, in

growth cabinets maintained at 22oC with 30-40% humidity and a light intensity of ~115

µmol m2s1. For vernalization treatment Medicago seed was first scarified, germinated

overnight at 22oC on moist petri dishes and placed at 4oC in the dark for 2 weeks. For

cultivation of Medicago plants soil composed of a mixture of 9 parts Black Magic® seed

raising mix (Yates, Orica New Zealand Ltd.), 3 parts coarse-graded vermiculite (Pacific

Growers Supplies Ltd.) and 1 part No. 2 Propagating Sand (Daltons Ltd.). After one

month of growth, watering was supplemented with hydroponics media (without

Na2SiO3)(Gibeaut et al., 1997). For in vitro culture, M. truncatula var Jester or R108

seeds were scarified, sterilized and cold treated for 3 d or 2 weeks (V) at 4 oC.

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

with Kalys agar HP 696-7470 (Kalys, France) in Magenta boxes (Sigma). Flowering time

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

(when the first fully open 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 transformation vector, pB2GW7

(Karimi et al., 2002). Agrobacterium strain GV3101 (Koncz and Schell, 1986) containing

the pB2GW7 vectors were applied to Arabidopsis ft-1 mutant flowers using the protocol

described by Martinez-Trujillo et al. (2004). Seed was sown directly onto soil for

selection for transformants using the Basta herbicide. Putative transformants were

confirmed by either gDNA PCR or qRT PCR analysis. A minimum of ten transformants

were characterized for each transgene

Transformation of Medicago R108 with Agrobacterium and regeneration via somatic

embryogenesis



27

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 plants growing in

vitro in LDs without vernalization were co-cultivated with Agrobacterium containing the

constructs and regenerated via somatic embryogenesis according to the Medicago

truncatula handbook (Cosson et al., 2006). Selection for transformant plants was with

Basta (3 mg/L, Kiwicare) or DL-phosphinotricin (2 mg/L or 3 mg/L, Sigma). Explants

without Agrobacterium infiltration went through somatic embryogenesis on selective or

non-selective media as negative control or regeneration control, respectively.

Regenerated T0 transformed plantlets were further cultivated in soil.

RNA extraction, cDNA synthesis and qRT-PCR

RNA was extracted from ~100 mg of plant tissue using either the RNeasy® Plant Mini

Kit (Qiagen) followed by TURBO DNase on-column treatment (TURBO DNA-freeTM

Kit, Applied Biosystem), or using RNA purification reagent (Invitrogen) and subsequent

DNase treatment (Invitrogen) as per manufacturer’s instructions. Total RNA (0.5-1µg)

was transcribed into cDNA with Superscript III reverse transcriptase (Invitrogen) using

the (dT)17 primer (Frohmann 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 Applied Biosystems 7900HT Sequence Detection

System. For the Roche LC480 qRT-PCR was performed in 10µL reaction volumes using

the LightCycler 480 SYBR Green I Master (Roche). cDNA was diluted 30-fold and 3µL

used in each reaction. Final primer concentrations were 0.5µM and all primer

combinations showed efficiencies greater than 1.8 using an annealing temperature of

58oC. For qRT-PCR performed using the AB 7900HT Sequence Detection System, 2μL

of a 20-fold diluted solution of cDNA sample was used in a total reaction volume of

10μL 1x SYBR® Green PCR Master Mix (Applied Biosystems) with final primer

concentrations of 0.5μM. Relative gene expression levels were calculated using the

ΔΔCT method (Livak and Schmittgen, 2001) with modifications (Bookout and



28

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 5000

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 sequenced. 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 accession numbers: MtFTa1 gDNA AC123593 (Mt3.5; Medtr7g084970.1,

chr7:25432978..25435370); MtFTa2 gDNA AC123593 (Medtr7g085020.1); MtFTc

gDNA AC123593 (Mt3.5, Medtr7g085040.1, chr7:25463178..25466505); MtFTb1

gDNA AC167329 (Mt3.5, Medtr7g006630.1, chr7:979293..981445); MtFTb2 gDNA

AC167329 (Mt3.5, Medtr7g006690.1, chr7:1019320..1022742), MtFTa1 cDNA

HQ721813; MtFTa2 cDNA HQ721814; MtFTb1 cDNA HQ721815; MtFTb2 cDNA

HQ721816 and MtFTc cDNA HQ721817

ACKNOWLEDGEMENTS

We thank Jane Campbell and Robyn Lough for technical assistance.



29

FIGURE LEGENDS

Figure 1. Three classes of FT-like proteins are present in legumes (Fabales order)

but not in other Rosid orders

A, Phylogram of FT-like proteins sequences from different Rosid orders. The legume

FTa, b and c groups are indicated. B, Partial amino acid alignment of FT sequences and

other PEBP proteins. Asterisks on top row indicate Tyr85(Y)/His88(H) and

Gln140(Q)/Asp144(D) residues distinguishing FT-like and TFL1-like members. 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 the Geneious

software. The sequences are from; Arabidopsis (Arabidopsis thaliana, At), Sugar beet

(Beta vulgaris, Bv). Orange (Citrus unshiu, Ci), pumpkin (Cucurbita maxima, Cm), fig

(Ficus carica, Fc), woodland strawberry (Fragaria vesca, Fv), soybean (Glycine max),

cotton (Gossypium hirsutum, Gh), apple (Malus x domestica, Md), Medicago (Medicago

truncatula, Mt), rice (Oryza sativa, Os), Lombardy poplar (Populus nigra, Pn), trifoliate

orange (Poncirus trifoliate, Pt), Japanese apricot (Prunus mume, Pm), pea (Pisum

sativum, Ps), Asian pear (Pyrus pyrifolia, Py), China rose (Rosa chinensis Rc), tomato

(Solanum lycopersicum, Le) and grape (Vitis vinifera Vv).

Figure 2. Expression of the FT-like genes in different tissues

Medicago truncatula cv. Jester plants grown for 15-day (A) and 35-day (B) under LD

after 2 weeks vernalization at 4 °C in the dark. Arrows indicate the tissues harvested and

representative age of plants to investigate expression. Harvesting was done at 2 h after

dawn (ZT 2). C, Transcription profiles of MtFTa1, FTa2, FTb1, FTb2 and FTc in various

tissue types as shown in A and B. The average +/- S.E. of three biological replicates is

shown for each sample and transcripts were normalized to PDF2 (protodermal factor 2).

Figure 3 MtFTs have different expression patterns during development

A, to F, Relative transcript profiles over a time course of whole seedling (FTa1, FTa2,

FTb1 and FTb2) or the uppermost dissected apical buds (FTc and MtPIM). Medicago

truncatula var. Jester seedlings were vernalized for 2 weeks in the dark at 4 °C and grown



30

under LDs for up to 25 days. Harvesting was performed at ZT 2. MtPIM was used as a

marker of the floral transition. A flower bud had emerged from the node or visible on

whole plants harvested at 25 days (5th or 6th node). The average +/- S.E. between three

biological replicates are shown and transcripts were normalized to PDF2.

Figure 4 Vernalization and LD photoperiod promote flowering and MtFTa1

expression

A, Flowering time of Medicago truncatula cv. Jester plants vernalized in the dark at 4 °C

for 2 weeks (V) or non-vernalized (NV) and grown in either LD (16 h light: 8 h dark) or

SD (8 h light: 16 h dark) photoperiod. Flowering time was expressed as either number of

days to produce the first flower (days to first flower) or the node from which the first

flower emerged (nodes to first flower). Nodes were counted from the base of the plant to

the growing tip of the primary stem. The data represents an average +/- S.E. of ten plants.

B, MtFT transcript levels (determined using qRT-PCR) in whole seedlings grown under

LD with or without vernalization. C, MtFT transcript levels in whole seedlings vernalized

and grown in LD or SD. Seedlings were harvested with 3 nodes at 15-days old (LD) or

21-days old (SD) at ZT 2. The data represents an average +/- S.E. of three biological

replicates, with transcripts normalized to PDF2.

Figure 5 Photoperiodic regulation of MtFT gene expression

A, Relative transcript levels of MtFT genes was measured every 4 h through the diurnal

cycle in long (LD) and short day (SD) conditions using qRT-PCR. LDs were 16 h light:

8 h dark and SDs were 8 h light: 16 h dark. ZT0 is lights on. The graphs show i, MtFTa1,

ii MtFTa2, iii MtFTb1, iv MtFTc transcript levels in LD and SD and iv, the above genes

in LD, and v, the above genes in SD. Below graphs v and vi, the open bars indicate day,

black bars indicate night. Transcript abundance of the MtFT genes was normalized to

TEF1a and calibrated to the sample with the highest gene expression (FTb1 at ZT 4 in

LD). Total aerial parts of vernalized 10 d Jester plants grown in vitro were used. The

average +/- S.E. of qRT PCR on 4 biological replicates is shown. B, i, Flowering time

(node to first flower) of Medicago truncatula cv. Jester plants shifted from SDs to LDs

for different durations. ‘LD only’ and ‘SD only’ refer to non-shifted control plants.



31

Plants to be shifted were grown under SD until the monofoliate leaf unfolded (~5-7

days). Plants were then shifted to LDs for the duration of the experiment or for 1, 3, 5 and

8 days before transfer back into SDs. Shifts between LDs and SDs occurred at dawn;

therefore plants received a 16h (shift to LD) or 8h (shift to SDs) light period before night.

Data represents the average +/- S.E. for ten plants. B, ii-iv, A transcript profile of FTa1,

FTa2 and FTb1 during shifts between SDs and LDs. Whole plants to be shifted were

grown under SDs until the monofoliate leaf unfolded. A subset of SD plants were

harvested and the remaining plants shifted into LDs. Additional groups of plants moved

into LDs were harvested after 1, 3 and 5 LDs and those remaining were shifted back to

SDs. SD grown plants were harvested after a further 1, 3 and 5 days. Seedlings were

harvested at ZT2 irrespective of LDs or SDs. The data represents an average +/- S.E. of

three biological replicates, with transcripts normalized to β-tubulin.

Figure 6 Overexpression of MtFTa1, FTb1 and FTc complement the Arabidopsis ft-1

mutation

A, Photograph of the 35S:MtFTs lines in the Arabidopsis ft-1 mutant (Ler). Ectopic

expression of MtFTa1, MtFTb1 and MtFTc resulted in early flowering of ft-1 mutant

plants. B, A representative line expressing each MtFT construct was selected for

flowering time analysis. Total leaf number was calculated by combining total rosette

leaves and cauline leaves on the primary inflorescence. Data represents a minimum of 10

plants scored for each line +/- S.E. MtFTa1 and MtFTc overexpression lines were

consistently early flowering (greater than 10 independent lines scored), whereas MtFTb1

transformants displayed early and intermediate flowering times (Supplemental Figure 4).

Figure 7 Overexpression of FTa1 in Medicago truncatula results in early flowering

A, Flowering time of Medicago truncatula cv. R108 transformed with 35S:FTa1 or

35S:GUS constructs. Regeneration controls (RC) are plants that were regenerated from

leaf explants in the absence of PPT selection. The graph shows flowering times of T0

transformants and control plants in LD conditions without vernalization, expressed as

nodes to first flower. The data from 9 independent 35S:FTa1 transformants, 5

independent 35S:GUS lines and 8 RC plants was used to calculate the mean number of



32

nodes to flowering +/- S.E. B, MtFTa1 transcript accumulation in T0 transformants and

control plants was measured in LD conditions using qRT-PCR. Relative transcript

abundance of MtFTa1 in fully expanded trifoliate leaves at ZT1.5 is shown for 3

independent 35S:FTa1 plants, one 35S:GUS plant and one RC plant. Data represents the

average +/- S.E. of 2 biological replicates, with transcripts normalized to TEF1a. C, T0

transformant and control plants grown under LDs. Photographs were taken 42 to 44 days

after transfer of regenerated plantlets to soil. The white arrow indicates a flower. D and E,

Graphs showing the days to first flower (D) and nodes to first flower (E) of 35S:FTa1 T1

and R108 control plants in LD conditions without vernalization. The flowering time of

the progeny of two independent T0 transformants was measured. All the progeny of M1-

4 flowered early (n=12), while the progeny of M18-2 showed segregation with 2 plants

flowering early and 1 plant flowering as late as the R108 control plants (n=10).

Figure 8 ftla1 mutants are late flowering

A, Schematic of the MtFTa1 gene with the position of fta1-1 (NF3307; +56) and fta1-2

(NF2519; +273) marked. Exons are shown by black boxes and thin lines represent

introns. +1 refers to the A of ATG. B, Photograph of R108 and an fta1-1 mutant plant

taken 35 d after germination. R108 is producing flowers (arrows). Plants were

vernalized for 2 weeks and then grown in LDs. C, A graph showing the relative

expression of MtFTa1 in R108 (wild type) and plants homozygous for the fta1-1or fta1-2

mutation. RNA was extracted from fully expanded trifoliate leaves grown under LDs at

15 d after germination, following a vernalization treatment. Data represents the average

relative expression for 3 biological replicates +/- S.E. D, A comparison of flowering

times (measured using days to first flower) of R108 and fta1-1 plants in different growth

conditions (LDs or SDs), vernalized (V) or non-vernalized (NV). Data represents the

average of at least 6 plants +/- S.E. E, The relative expression of MtFTa1 in R108 grown

in LDs (non-vernalized). The uppermost fully expanded trifoliate leaf was harvested

from plants with 4, 6-8, 14-16, and 18+ nodes. Plants with 18+ nodes had flowered. Data

represents the average for 3 biological replicates +/- S.E.



33

Figure 9 Altered expression of Medicago FTs and floral meristem identify genes in

the fta1-1 mutant

A, to D, Relative transcript levels of FTs and the meristem identify genes, MtPIM and

MtFULc in wild-type R108 and the fta1-1 mutant in leaf or dissected apical bud tissue

from Medicago plants ~10 days old with 2 nodes (2n), ~25 days old with 5 nodes (5n) or

once the plants had flowered. Plants were vernalized for 2 weeks in the dark at 4 °C and

grown under LDs. Tissue was harvested at ZT2. The data represents an average +/- S.E.

of three biological replicates, with transcripts normalized to PDF.

Figure 10 Wild type flowering of ftlc mutants caused by Tnt1 insertions

A, Schematic showing the position of Tnt1 insertions in the MtFTc gene. Using a reverse

genetics approach Tnt1 insertions were identified in the 5’ UTR (NF6335; ftc1), exon 1

(NF4345; ftc2) and intron 1 (NF4913; ftc3). B, Flowering time of ftc mutants in

vernalized LD plants. Data represents the average of 8 plants homozygous for the Tnt1

insertion +/- S.E. C, and D, qRT PCR of MtFTc and MtPIM - flowers



34

SUPPLEMENTAL DATA

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

Supplemental Figure S1. Genomic structure 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 diurnal expression profile of MtFT

genes in LD and SD conditions.

Supplemental Figure S4. Photoperiodic regulation of MtFT gene expression.

Supplemental Figure S5. Flowering time and transgene expression levels in ten

Arabidopsis ft-1 lines expressing MtFTb1 or MtFTb2.

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

Supplemental Figure S7. Characterization of the fta1 mutant alleles.

Supplemental Figure S8. Altered expression of Mediago 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.



35

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A

B

C

Faba

le F

Tsot

her R

osid

FTs

othe

r FTs

TFL

BFT

MFT

* *Segment B

Fabales

FTa

FTb

FTc

Brassicales

Vitales

CucurbitalesMalpighiales

Sapindales

Rosales

Malvales

A B



Monofoliateleaf

Root

Cotyledon

Expandedtrifoliateleaf

Foldedtrifoliateleaf

Stem

Flower

Flower bud

Seedpod

Apical buds

A

B

0.8

0.6

0.4

0.2

0

MtFTa1

Roo

t

Cot

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ons

Fold

ed tr

ifolia

te le

af

Mon

ofol

iate

leaf

Exp

ande

d tri

folia

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af

Flow

er b

uds

Flow

ers

See

d po

d

Ste

m

Api

cal b

uds

0.008

0.006

0.004

0.002

0

MtFTc

Exp

ress

ion

rela

tive

to P

DF2

0.15

0.1

0.05

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Figure 2. Expression of the MtFT genes in different tissues

Medicago truncatula cv. Jester plants grown for 15-day (A) and 35-day (B) under LD after 2 weeks vernalization at 4 °C in the dark. Arrows indicate the tissues harvested and representative age of plants to investigate expression. Harvesting was done at 2 h after dawn (ZT 2). C, Transcription pro�les of MtFTa1, FTa2, FTb1, FTb2 and FTc in various tissue types as shown in A and B. The average +/- S.E. of three biological replicates is shown for each sample and transcripts were normalized to PDF2 (protodermal factor 2).



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Figure 3. MtFT genes have different expression patterns during development

A, to F, Graphs showing relative transcript profiles over a time course of 25 days (from germi-nation) in whole seedling extracts (FTa1, FTa2, FTb1 and FTb2) or the uppermost dissected apical buds (FTc and MtPIM). Harvesting was performed at ZT 2. Medicago truncatula var. Jester seedlings were vernalized for 2 weeks in the dark at 4 °C and grown under LDs. MtPIM was used as a marker of the floral transition. A flower bud had emerged from the node or visible on whole plants harvested at 25 days (5th or 6th node). The average and standard error between three biological replicates is shown and transcripts were normalized to PDF2.



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Figure 4 Vernalization and LD photoperiod promote flowering and MtFTa1 expression A, Flowering time of Medicago truncatula cv. Jester plants vernalized (V) or non-vernalized (NV) and grown under LDs or SDs. Vernalization was in the dark at 4 °C for 2 weeks. Flowering time was expressed as either number of days to produce the first flower (days to first flower) or the node from which the first flower emerged (nodes to first flower). Nodes were counted from the base of the plant to the growing tip of the primary stem. The data represents an average and standard error of ten plants.B & C, MtFT transcript levels (determined using qRT-PCR) in whole seedlings grown under LD or SD, with (+V) or without (-V) a vernalization treatment. Seedlings were harvested with 3 nodes at 15-days old (LDs) or 21-days old (SDs) at ZT 2. The data represents an average and standard error of three biological repli-cates, with transcripts normalized to PDF2.

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Figure 5 Photoperiodic regulation of MtFT gene expression A, Relative transcript levels of MtFT genes was measured every 4 h through the diurnal cycle in long (LD) and short day (SD) conditions using qRT-PCR. LD is photoperiods (16 h light: 8 h dark) and SD is photoperiods (8 h light: 16 h dark). ZT0 is lights on. The graphs shown i, MtFTa1, ii MtFTa2, iii MtFTc, iv MtFTb1 transcript leves in LD and SD and iv, the above genes in LD, and v, the above genes in SD. Below graphs v and vi, the open bars indicate day, black bars indicate night. Transcript abundance of the MtFT genes was normalised to TEF1a and calibrated to the sample with the highest gene expression (FTb1 at ZT 4 in LD). Total aerial parts of vernalization Jester plants with two fully-expanded trifoliate leaves, grown in vitro, were used. The mean +/- SE of qPCR on 4 biological replicates is shown. B, i, Flowering time (node to first flower) of plants shifted from SDs to LDs for varying lengths of time. ‘LD only’ and ‘SD only’ refer to non-shifted control plants. Plants to be shifted were grown under SD until the monofoliate leaf unfolded (~5-7 days). Plants were then shifted to LDs for the duration of the experiment or for 1, 3, 5 and 8 days before transfer back into SDs. Shifts LDs and SDs occurred at dawn; therefore plants received a 16h (shift to LD) or 8h (shift to SDs) light period before night. Data represents the average and standard error for ten plants.ii-iv, A transcript profile of FTa1, FTa2 and FTb1 during shifts between SDs and LDs. Whole plants to be shifted were grown under SDs until the monofoliate leaf unfolded. A subset of SD Plants was harvested at and the remaining shifted into LDs. Additional groups of plants moved into LDs were harvested at after 1, 3 and 5 LDs and those remaining shifted back to SDs. SD grown plants were harvested after a further 1, 3 and 5 days. Seedlings were harvested at ZT2 irrespective of LDs or SDs. The data represents an average and standard error of three biological replicates, with transcripts normalized to β-tubulin.

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Figure 6 Overexpresion of MtFTa1, MtFTb1 and MtFTc complement the Arabidopsis ft-1 mutation

A, Photograph of the 35S:MtFTs lines in the Arabidopsis ft-1 mutant (Ler). Ecto-pic expression of MtFTa1, MtFTb1 and MtFTc resulted in early flowering of ft-1 mutant plants.B, A representative line expressing each MtFT construct was selected for flower-ing time analysis. Total leaf number was calculated by combining total rosette leaves and cauline leaves on the primary inflorescence. Data represents a minimum of 10 plants scored for each line +/- the standard error. MtFTa1 and MtFTc overexpression lines were consistently early flowering (greater than 10 independent lines scored), whereas MtFTb1 lines displayed early and intermedi-ate flowering times (Supplemental Figure 4).



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A, Flowering time of Medicago truncatula R108 transformed with 35S:FTa1 or 35S:GUS gene expression constructs. Regeneration controls (RC) are plants that were regenerated from leaf explants in the absence of PPT selection. The graph shows flowering times of T0 transformants and control plants in NV LD condi-tions, expressed as nodes to first flower. The data from 9 independent 35S:FTa1 transformants, 5 indepen-dent 35S:GUS lines and 8 RC plants was used to calculate the mean floral node and standard error. B, MtFTa1 transcript accumulation in T0 transformants and control plants was measured in LD conditions using qRT-PCR. Relative transcript abundance of MtFTa1 in fully expanded trifoliate leaves at ZT1.5 is shown for 3 independent 35S:FTa1 plants, one 35S::GUS plant and one RC plant. Data represents the average and standard error of 2 biological replicates, with transcripts normalized to TEF1a.C, T0 transformant and control plants grown under LDs. Photographs were taken 42 to 44 days after trans-fer of regenerated plantlets to soil. The white arrow indicates a flower. D and E, Graphs showing the days to first flower (D) and nodes to first flower (E) of 35S:FTa1 T1 and R108 control plants in LD-V conditions. The flowering time of the progeny of two independent T0 transformants was measured. All the progeny of M1-4 flowered early (n=12), while the progeny of M18-2 showed segrega-tion with 2 plants flowering early and 1 plant flowering as late as the R108 control plants (n=10).



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Figure 8 fta1 mutants are late �owering A, Schematic of the MtFTa1 gene with the position of fta1-1 (NF3307; +56) and fta1-2 (NF2519; +273) marked. Exons are shown by black boxes and thin lines represent introns. +1 refers to the A of ATG. B, A photograph of R108 and an fta1-1 mutant plant taken 35 days after germination. R108 is producing �owers (arrows). Plants were vernalized for 2 weeks and then grown in LDs. C, A graph showing the relative expression of MtFTa1 in R108 (wild-type) and plants homozygous for the fta1-1or fta1-2 mutation. RNA was extracted from fully expanded trifoliate leaves grown under LDs at 15d after germination, following a vernalization treatment. Data represents the average relative expression for 3 biological replicates +/- S.E. D, A comparison of �owering times (measured using days to �rst �ower) of R108 and fta1-1 plants in di�erent growth conditions (LDs or SDs), with (V) or without (NV) a vernalization treatment. Data represents the average of at least 6 plants +/- standard error. E, The relative expression of MtFTa1 in R108 grown in LDs (not vernalized). The uppermost fully expanded trifoliate leaf was harvested from plants with 4, 6-8, 14-16, and 18+ nodes. Plants with 18+ nodes had �owered. Data represents the average for 3 biological replicates +/- standard error.

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Figure 9 Altered expression of Medicago FTs and floral meristem identify genes in the fta1-1 mutant

A, to D, Relative transcript levels of FTs and the meristem identify genes, MtPIM and MtFULc in wild-type R108 and the fta1-1 mutant in leaf or dissected apical bud tissue from Medicago plants ~10 days old with 2 nodes (2n), ~25 days old with 5 nodes (5n) or once the plants had flowered (~30 days old for R108 and ~90 days old for fta1-1). Plants were vernalized for 2 weeks in the dark at 4 °C and grown under LDs. Tissue was harvested at ZT2. The data represents an average +/- S.E. of three biological replicates, with tran-scripts normalized to PDF.

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Figure 10 Wild type flowering of ftc mutants caused by Tnt1 insertions

A, Schematic showing the position of Tnt1 insertions in the MtFTc gene. Using a reverse genetics approach Tnt1 insertions were identified in the 5’ UTR (NF6335; ftc-1), exon 1 (NF4345; ftc-2) and intron 1 (NF4913; ftc-3).B, Relative MtFTc transcript levels (normalized to PDF) in the wild-type and the three ftc mutant alleles.C, Flowering time of ftc mutants in LD V plants. Data represents the average of 8 plants homozygous for the Tnt1 insertion +/- S.E.



FT regulation of Medicago flowering Corresponding Author: NEW … · production of a mobile floral...

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Transcript of FT regulation of Medicago flowering Corresponding Author: NEW … · production of a mobile floral...