Isolation and Functional Characterization of MsFTa, a FLOWERING LOCUS T Homolog from Alfalfa (Medicago sativa)

The production of hay and seeds of alfalfa, an important legume forage for the diary industry worldwide, is highly related to flowering time, which has been widely reported to be integrated by FLOWERING LOCUS T (FT). However, the function of FT(s) in alfalfa is largely unknown. Here, we identified MsFTa, an FT ortholog in alfalfa, and characterized its role in flowering regulation. MsFTa shares the conserved exon/intron structure of FTs, and the deduced MsFTa is 98% identical to MtFTa1 in Medicago trucatula. MsFTa was diurnally regulated with a peak before the dark period, and was preferentially expressed in leaves and floral buds. Transient expression of MsFTa-GFP fusion protein demonstrated its localization in the nucleus and cytoplasm. When ectopically expressed, MsFTa rescued the late-flowering phenotype of ft mutants from Arabidopsis and M. trucatula. MsFTa over-expression plants of both Arabidopsis and M. truncatula flowered significantly earlier than the non-transgenic controls under long day conditions, indicating that exogenous MsFTa strongly accelerated flowering. Hence, MsFTa functions positively in flowering promotion, suggesting that MsFTa may encode a florigen that acts as a key regulator in the flowering pathway. This study provides an effective candidate gene for optimizing alfalfa flowering time by genetically manipulating the expression of MsFTa.


Introduction
Flowering time, the switch from vegetative to reproductive growth, is a key trait for the seasonal and geographical adaptation of flowering plants. The mechanism of flowering is best understood in the annual plant Arabidopsis, in which the timely onset of flowering is regulated by an intricate network with various genetic regulators responding to endogenous (gibberellin, autonomous and aging) or environmental (vernalization and photoperiod) triggers, as reviewed in recent years [1][2][3][4]. The balance of signals from these pathways is integrated by a common set of genes, including FLOWERING LOCUS C (FLC)/MADS AFFECTING FLOWEIRNG (MAF), FLOWERING LOUCS T (FT), CONSTANS (CO) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), to determine the flowering time [5][6][7]. In Medicago, however, FLC/MAF clade flowering repressors are missing and CO-like genes do not seem to regulate flowering [8][9][10]. Hence, florigen encoding gene FT, acting as a long-distance hormone signal between leaves and shoot apical meristems, is the most important flowering integrator of the legume species.
Since the search for the flowering hormone florigen in the 1930s, an increasing number of FT-like genes have been identified in a wide range of annual plants, such as rice, soybean and barrel clover [11][12][13]. For example, in M. truncatula five FT-like genes, namely MtFTa1, MtFTa2, MtFTb1, MtFTb2 and MtFTc, have been characterized. Among them, MtFTa1, MtFTb1 and MtFTc rescued the late flowering phenotype of Arabidopsis ft-1 mutant, while the other two genes failed to act as a florigen [11]. Functional differentiation among FT paralogs derived from lineage-specific duplication has been observed in several species, such as soybean, potato and rice [13][14][15]. The diversity of the functions of FT paralogs is attributed to the expression pattern shift and the divergence in gene regulatory networks.
In recent years, studies on FT-integrated flowering regulation have focused on economically important perennial plants including grasses (e.g., switchgrass and ryegrass) [16,17] and trees (e.g., poplar, apple and cherry) [18][19][20]. In contrast to annual plants, FT is expressed in both leaves and flowers of several perennial species such as sweet cherry, kiwifruit and leafy spurge [21][22][23]. Functional coordination of perennial FT paralogs revealed the evolution of a complex perennial adaptive trait after genome duplication. For example, poplar FT1 determined the onset of reproductive stage in winter, whereas FT2 promoted vegetative growth and inhibited bud set in the growing season [18]. Ectopic over-expression of FT perennial orthologs in Arabidopsis caused early flowering in most cases, suggesting a conserved function in perennial species [16][17][18][19][20]22].
Alfalfa (M. sativa), one of the most important forages globally, exhibits seasonal flowering. Different from perennial woody plants, which are not able to form flower buds during the first several years of their life cycle, herbaceous alfalfa grows vegetatively at early developmental stages and then switches to the reproductive stage in the same year. In Northern China, a main alfalfa planting region in the country, the first cutting coincides with the rain season, which severely limits hay production. Breeding alfalfa cultivars with optimal flowering time would facilitate the improvement of the quantity and quality of alfalfa. Our study here demonstrated that MsFTa, the first isolated FT from alfalfa, not only shared the conserved functional domains of its orthologs, but also acted ectopically in accelerating flowering. These findings would supply a potential candidate for the generation of alfalfa cultivars with an optimal flowering time for the main planting regions in Northern China via genetic modulation of MsFTa expression.
To investigate flowering regulation in perennial legume alfalfa, we cloned an FT-like cDNA from the forage by combination of RT-PCR and RACE strategy based on the sequence information of MtFTs. The 1029 bp cDNA sequence contained an open reading frame (ORF) of 531 bp (GenBank: JF681135) ( Figure S1). The putative protein consists of 176 amino acids with theoretical isoelectric point (pI) of 8.23 and molecular weight of 19.7 kDa, respectively. Phylogenetic analysis of 33 FT and FT-like proteins from rice, barley, Arabidopsis, soybean, barrel clover and poplar demonstrated that the six MtFTs were grouped into three subclasses, namely Subclass A (MtFTa1, MtFTa2 & Mt6g033040), Subclass B (MtFTb1 & MtFTb2) and Subclass C (MtFTc) (Figure 1). The putative alfalfa FT protein was clustered into Subclass A sharing the same branch with MtFTa1 and MtFTa2 ( Figure  1). Hence, it was designated as MsFTa, the first FLOWERING LOCUS T gene identified in alfalfa. Compared with Subclass A members from Arabidopsis, rice and barley, MsFTa was more closely related to the FTs of legume plants such as barrel clover and soybean ( Figure 1). Sequence homology analysis showed that MsFTa shared an identity of 59-99% with MtFT members, the lowest identity with MtFTc and the highest with MtFTa1, respectively (Table S1). The sequence similarity of MsFTa with FTs from the other five species ranges from 54% to 84% (Table S1). Multiple alignment revealed that, like its orthologs, MsFTa has a ligand-binding motif with a conserved key residue Tyr (Y) at position 85, an external loop of a 14-amino acid segment (residues 128-141) and a triad (residues 150- ; Glycine max (Gm): Gm19g108100, Gm18g298900, Gm18g299000, Gm16g151000, Gm16g044200, Gm16g150700, Gm16g044100, Gm19g108200, Gm08g363100; Populus trichocarpa (Pt): Pt8g077700, Pt10g179700; Hordeum vulgare (Hv): Hv1Hr1g076430, Hv2Hr1g023180, Hv2Hr1g084540, Hv3Hr1g027590, Hv3Hr1g087100, Hv4Hr1g012200, Hv7Hr1g024610 and Oryza sativa (Os): OsHd3a (Os06g0157700), Os04g0488400, Os05g051800, Os06g0157500, Os06g0552900, Os11g0293800, Os12g0232300.
To investigate flowering regulation in perennial legume alfalfa, we cloned an FT-like cDNA from the forage by combination of RT-PCR and RACE strategy based on the sequence information of MtFTs. The 1029 bp cDNA sequence contained an open reading frame (ORF) of 531 bp (GenBank: JF681135) ( Figure S1). The putative protein consists of 176 amino acids with theoretical isoelectric point (pI) of 8.23 and molecular weight of 19.7 kDa, respectively. Phylogenetic analysis of 33 FT and FT-like proteins from rice, barley, Arabidopsis, soybean, barrel clover and poplar demonstrated that the six MtFTs were grouped into three subclasses, namely Subclass A (MtFTa1, MtFTa2 & Mt6g033040), Subclass B (MtFTb1 & MtFTb2) and Subclass C (MtFTc) (Figure 1). The putative alfalfa FT protein was clustered into Subclass A sharing the same branch with MtFTa1 and MtFTa2 ( Figure 1). Hence, it was designated as MsFTa, the first FLOWERING LOCUS T gene identified in alfalfa. Compared with Subclass A members from Arabidopsis, rice and barley, MsFTa was more closely related to the FTs of legume plants such as barrel clover and soybean ( Figure 1). Sequence homology analysis showed that MsFTa shared an identity of 59-99% with MtFT members, the lowest identity with MtFTc and the highest with MtFTa1, respectively (Table S1). The sequence similarity of MsFTa with FTs from the other five species ranges from 54% to 84% (Table S1). Multiple alignment revealed that, like its orthologs, MsFTa has a ligand-binding motif with a conserved key residue Tyr (Y) at position 85, an external loop of a 14-amino acid segment (residues 128-141) and a triad (residues 150-152) at the C-terminal end ( Figure 2) [24][25][26]. In addition, MsFTa features a large number of alpha helices, beta-folds and random coil structures ( Figure S2a). Our prediction using ScanProsite showed that most regions of MsFTa are hydrophilic ( Figure S2b), indicating it is a hydrophilic protein. 152) at the C-terminal end ( Figure 2) [24][25][26]. In addition, MsFTa features a large number of alpha helices, beta-folds and random coil structures ( Figure S2a). Our prediction using ScanProsite showed that most regions of MsFTa are hydrophilic ( Figure S2b), indicating it is a hydrophilic protein. Comparison of the exon-intron structure of FT-like genes demonstrated that like most FT-like genes from a variety of species [11,13,27,28], MsFTa is composed of four exons separated by three introns (Figure 3). Interestingly, exon 2 is universally 62 bp and exon 3 is 41 bp, except two barley FTlike genes (Hv3Hr1g0871002 and Hv7Hr1g0246102) ( Figure 3). The 2nd and 3rd exons of MsFTa encode the N-terminal of the ligand-binding motif, including the amino acids forming the ligandbinding pocket and the key residue Tyrosine (Y) at position 85 ( Figure 2). Therefore, MsFTa not only Comparison of the exon-intron structure of FT-like genes demonstrated that like most FT-like genes from a variety of species [11,13,27,28], MsFTa is composed of four exons separated by three introns

The MsFTa-GFP Recombinant Protein Resided in Both the Cytoplasm and Nucleus
To examine the subcellular localization of MsFTa, 35S::MsFTa-GFP and 35S::GFP were transformed into onion epidermal cells separately. The transient expression of the GFP control was uniformly distributed throughout the cell, whereas the MsFTa-GFP fusion protein was observed in the nucleus and cytoplasm, especially the membrane ( Figure 4). This result is consistent with the recent observations of FT-like proteins in perennial species such as switchgrass and sweet cherry [16,22].

The MsFTa-GFP Recombinant Protein Resided in Both the Cytoplasm and Nucleus
To examine the subcellular localization of MsFTa, 35S::MsFTa-GFP and 35S::GFP were transformed into onion epidermal cells separately. The transient expression of the GFP control was uniformly distributed throughout the cell, whereas the MsFTa-GFP fusion protein was observed in the nucleus and cytoplasm, especially the membrane ( Figure 4). This result is consistent with the recent observations of FT-like proteins in perennial species such as switchgrass and sweet cherry [16,22].

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction in MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested.
MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerates Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction of MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested. MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold of the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Diurnal and Tissue-Specific Expression Pattern of MsFTa
To monitor the temporal and spatial expression profiles of MsFTa, quantitative RT-PCR was performed using MsFTa-specific primers. Analysis of the MsFTa transcript demonstrated that the level of MsFTa progressively increased after 6 h light and reached a peak at 16 h light. The onset of darkness caused a sharp deduction in MsFTa and the descending trend continued during the night period ( Figure 5a). Spatially, four tissues from both underground and aerial organs were tested.
MsFTa was detected in all tested tissues including roots, stems, floral buds and leaves with the highest expression in leaves, which is approximately 3.4-fold the amount in roots (Figure 5b). The expression pattern of MsFTa is highly reminiscent of its legume homologs, especially MtFTa2 in M. truncatula [11]. Detailed analysis demonstrated that besides cauline leaves, a higher level of FT transcript was detected in flowers, siliques, and seeds at early stages (http://bbc.botany.utoronto.ca/efp) ( Figure S3).

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerates Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with Bars represent the means ± SD of three biological replicates.

Ectopic Expression of MsFTa in Arabidopsis Strongly Accelerated Flowering
To explore the biological function of MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with kanamycin resistance was confirmed by PCR (Figure 6a). Semi-quantitative RT-PCR of the over-expression lines demonstrated that MsFTa transcript was abundantly expressed in both transgenic lines while absent in wild type Arabidopsis (Figure 6b). Phenotypically, the over-expression plants exhibited early flowering (Figure 6c). The measurement of leaf number showed that wild type plants flowered with 15.4 leaves, whereas T2-1 and T2-4 over-expression lines flowered with 12.1 and 11.4 leaves, respectively (p < 0.05) (Figure 6d). Consistently, in terms of days after germination (DAG), the first flower of line T2-1 and T2-4 appeared at 16.1 and 17.8 DAG, respectively, while the first flower of the wild type was observed at 24.3 DAG (p < 0.05) (Figure 6e). These results suggested that flowering time of the transgenic Arabidopsis was associated with MsFTa transcript level. kanamycin resistance was confirmed by PCR (Figure 6a). Semi-quantitative RT-PCR of the overexpression lines demonstrated that MsFTa transcript was abundantly expressed in both transgenic lines while absent in wild type Arabidopsis (Figure 6b). Phenotypically, the over-expression plants exhibited early flowering (Figure 6c). The measurement of leaf number showed that wild type plants flowered with 15.4 leaves, whereas T2-1 and T2-4 over-expression lines flowered with 12.1 and 11.4 leaves, respectively (p < 0.05) (Figure 6d). Consistently, in terms of days after germination (DAG), the first flower of line T2-1 and T2-4 appeared at 16.1 and 17.8 DAG, respectively, while the first flower of the wild type was observed at 24.3 DAG (p < 0.05) (Figure 6e). These results suggested that flowering time of the transgenic Arabidopsis was associated with MsFTa transcript level.

Ectopic Expression of MsFTa Promoted Flowering in Transgenic M. truncatula Plants
Given the close phylogenetic relationship and high sequence identity of MsFTa to MtFTa1, both barrel clover wild type R108 and mtfta1-1, a late-flowering mutant (NF3307) caused by the insertion of a Tnt1 retrotransposon in the first exon of MtFTa1 [11], were transformed with the construct of 35S::MsFTa mentioned above. Flowering time of the transgenic plants was measured as days to produce the first flower under LD conditions. Compared with R108 which flowered at D56, the two independent T1 lines (T1-1 & T1-2) overexpressing 35S::MsFTa produced the first flower at D35 and D32, respectively. Over-expression of MsFTa in M. truncatula resulted in statistically significant early flowering (p < 0.05) (Figure 7a). For mtft1-1 plants expressing 35S::MsFTa, the flowering time of two independent transgenic lines was analyzed. As shown in Figure 7b, the first flower of the two lines emerged at D58 and D61, while fta1-1 flowered at D83, which is significant later than R108 at D56 (p < 0.05), indicating that MsFTa successfully rescued the late flowering phenotype of fta1-1. These results suggested that similar to ectopic expression of MsFTa in Arabidopsis, introduction of MsFTa into M. truncatula caused strong flowering promotion.

Ectopic Expression of MsFTa Promotes Flowering in Transgenic M. truncatula Plants
Given the close phylogenetic relationship and high sequence identity of MsFTa to MtFTa1, both barrel clover wild type R108 and mtfta1-1, a late-flowering mutant (NF3307) caused by the insertion of a Tnt1 retrotransposon in the first exon of MtFTa1 [11], were transformed with the construct of 35S::MsFTa mentioned above. Flowering time of the transgenic plants was measured as days to produce the first flower under LD conditions. Compared with R108 which flowered at D56, the two independent T1 lines (T1-1 & T1-2) overexpressing 35S::MsFTa produced the first flower at D35 and D32, respectively. Over-expression of MsFTa in M. truncatula resulted in statistically significant early flowering (p < 0.05) (Figure 7a). For mtft1-1 plants expressing 35S::MsFTa, the flowering time of two independent transgenic lines was analyzed. As shown in Figure 7b, the first flower of the two lines emerged at D58 and D61, while fta1-1flowered at D83, which is significant later than R108 at D56 (p < 0.05), indicating that MsFTa successfully rescued the late flowering phenotype of fta1-1. These results suggested that similar to ectopic expression of MsFTa in Arabidopsis, introduction of MsFTa into M. truncatula caused strong flowering promotion. under LD conditions. Flowering time was measured as days to the emergence of the first flower under LD conditions. Two independent lines for each transformation were analyzed. Data represent average ± SD of three biological replicates. * indicates a significant difference between wild type and ft-10 (Student's t-test, p < 0.05).

Discussion
Precise flowering time ensures that the onset of flowering occurs at an ideal time to optimize plant biomass and fitness in specific environmental conditions. Although diverse upstream components of flowering pathways have been identified from plants, FLOWERING LOCUS T appears to be a universal regulator ultimately integrating flowering signaling in crops, vegetables, grass and trees [11,18,26,30]. In this study, we provided molecular and genetic evidence that MsFTa, an FT homolog from alfalfa, functions to accelerate flowering.
As a member of an ancient phosphatidylethanolamine-binding protein (PEBP) gene family, FT has a conserved exon/intron structure and highly identical protein sequence across species [13,31]. Indeed, our results demonstrated that MsFTa possessed the characteristic features of a classic FT gene. First, MsFTa, similar to FT-like genes, has four exons with conserved exon/intron boundaries [13,27,28]. Over-expression of the chimeric genes constructed by replacing individual exons of FT revealed that substitution of the fourth exon, especially the external loop caused late flowering, presumably the crystal structure was affected due to the sequence variation of the exon [24]. It has been reported recently that exons two and three were also crucial for the functional conversion of FT in Arabidopsis [13,32]. Second, MsFTa shared several well characterized amino acid residues proven to be important for flowering acceleration according to the experiments of individual residue exchange or segment replacement. For example, in Arabidopsis, alteration of Tyr 85 (Y85H) changed under LD conditions. Flowering time was measured as days to the emergence of the first flower under LD conditions. Two independent lines for each transformation were analyzed. Data represent mean ± SD of three biological replicates. * indicates a significant difference from wild type (Student's t-test, p < 0.05).

Discussion
Precise flowering time ensures that the onset of flowering occurs at an ideal time to optimize plant biomass and fitness in specific environmental conditions. Although diverse upstream components of flowering pathways have been identified from plants, FLOWERING LOCUS T appears to be a universal regulator ultimately integrating flowering signaling in crops, vegetables, grass and trees [11,18,26,30]. In this study, we provided molecular and genetic evidence that MsFTa, an FT homolog from alfalfa, functioned to accelerate flowering.
As a member of an ancient phosphatidylethanolamine-binding protein (PEBP) gene family, FT has a conserved exon/intron structure and highly identical protein sequence across species [13,31]. Indeed, our results demonstrated that MsFTa possessed the characteristic features of a classic FT gene. First, MsFTa, similar to FT-like genes, has four exons with conserved exon/intron boundaries [13,27,28]. Over-expression of the chimeric genes constructed by replacing individual exons of FT revealed that substitution of the fourth exon, especially the external loop caused late flowering, presumably the crystal structure was affected due to the sequence variation of the exon [24]. It has been reported recently that exons two and three were also crucial for the functional conversion of FT in Arabidopsis [13,32]. Second, MsFTa shared several well characterized amino acid residues proven to be important for flowering acceleration according to the experiments of individual residue exchange or segment replacement. For example, in Arabidopsis, alteration of Tyr 85 (Y85H) changed FT functions in promoting flowering, indicating that the absolutely conserved Tyr 85 is a key residue determining the protein function as an activator of flowering [25]. FTs share a triad ending with residue asparagine (N) at site 152, followed by leucine (Figure 2). The crystal structure revealed that the triplet motif contacts the adjacent external loop, which facilitates access to FT interactors, such as the bZIP transcription factor FD and 14-3-3 proteins [24,[33][34][35]. In Rosaceae species, mutations of the triad prevented the FT-like genes from promoting flowering [36]. Third, FTs have highly conserved motifs including a potential ligand-binding pocket and an external loop (Figure 2), both of which were proven to contribute to the functional specificity of FT [24,33]. Compared with other species, it seems that the external loop of legume plants is variant (Figure 2), implying varied conformation, possibly leading to flexible accessibility to a binding partner. Therefore, MsFTa, together with FT-like genes, specifies FT conformation by possessing identical exon/intron structure and highly conserved functional motifs.
FT-like orthologs from a variety of plant species have been shown to promote flowering when ectopically expressed [16,22,[37][38][39]. In the model legume M. truncatula, reverse genetics revealed that the loss-of-function mutant of MtFTa1, the closest homolog of MsFTa, caused late flowering and MtFTa1 fully complemented fta1-1 defects [11]. Consistently, an allelic series of gain-of-function mutations in MtFTa1 caused by retroelement insertions exhibited a dominant early flowering phenotype [40,41], confirming its function in flowering regulation. It appears that similar to FT, MsFTa from the perennial forage acts positively in flowering promotion. The notion is supported directly by the transgenic Arabidopsis and M. truncatula including both complementation and over-expression plants. Plants ectopically expressing MsFTa in either wild type or ft mutant of both plant species displayed a clear shift to early flowering (Figure 6d-g). Statistically, the flowering time difference between the transgenic plants and the non-transgenic ones is significant (p < 0.05). Notably, compared with wild type, complementation plants flowered slightly late, suggesting that constitutive expression of MsFTa partially rescued the flowering time of ft mutants from Arabidopsis and barrel clover. These findings imply a minute functional distinction between MsFTa and its orthologs, FT and MtFTa1, in Arabidopsis and barrel clover, respectively. Application of the endogenous promoter of MsFTa to produce transgenic alfalfa is undergoing. Moreover, generation of a loss of function mutant of MsFTa using the CRISPR-Cas9 strategy would be necessary to elucidate the endogenous function of MsFTa in alfalfa flowering regulation.
Given the detection of MsFTa in roots and stems, it is tempting to speculate that besides flowering promotion, MsFTa may participate in other development processes of alfalfa. FT orthologs have been documented to be involved in multiple functions like plant architecture [42], tuberization [15], and dormancy interruption [43]. The expression of MsFTa resembled FT orthologs in perennial plants, such as sweet cherry, loquat, leafy spurge and tea-oil tree, in which FT-like genes are expressed in diverse tissues, including mature leaves, floral buds and reproductive organs (flower organs and fruits) [21,22,27,44]. The expression divergence may be an important force in driving the functional division of FTs in perennial plants, especially in species with multiple FT paralogs [13,14,16,22]. As it is closely related to diploid legume M. truncatula, which has six identified FT-like genes, tetraploid alfalfa is more likely to have a similar number, or even more, MsFTa paralogs due to genome duplication. Long-term work of flowering regulation in alfalfa will be directed at paralogs of MsFTa.

Plant Material and Growth Conditions
Medicago sativa cv. 'Zhongmu No.1', bred by the Institute of Animal Science, the Chinese Academy of Agricultural Sciences, was used in this study. Seeds were germinated in regular soil in a 20 cm diameter pot. Medicago truncatula Tnt1 insertion mutant fta1-1 (NF3307) was obtained from the Noble Research Institute (Ardomor, OK, USA). Arabidopsis thaliana seeds of ft-10 (CS9869) were from Arabidopsis Biological Research Center (ABRC) and germinated in soil (vermiculite:soil = 1:3). All plants were grown in growth chambers at 21 • C under a 16/8 h (light/dark) photoperiod.

Cloning of MsFTa from Alfalfa
Total RNA was extracted from 2-week-old alfalfa seedlings with TRIzol reagent (Sangon, Shanghai, China). After DNase I (Fermentas-MBI, Shenzhen, China) treatment, the first-strand cDNA was synthesized using Reverse Transcriptase III (Takara, Dalian, China). A DNA fragment was amplified by using the two degenerate primers designed based on FT-like genes in M. truncatula and sequenced. The obtained partial sequence of alfalfa FT-like gene was used to design primers for amplification of the full-length MsFTa by 3 -RACE and 5 -RACE (Invitrogen, Carlsbad, CA. USA) with the specific primers according to the manufacturer's instructions. PCR amplicons were purified after separated on 1% agarose gel and cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced.

Bioinformatic Analysis
For FT homologous genes, Arabidopsis FT (At1g65480) was used as query sequence to search against the database EnsemblPlants (http://plants.ensembl.org/index.html). Phylogenetic analysis was performed with bootstrap trials of 1000 using DNAMAN version 7.0 (Lynnon Corporation, Quebec, QC, Canada), and sequence alignment was analyzed using the same software with default parameters. Isoelectric point (pI) and molecular weight (MW) values were predicted using ExPaSy (https:// prosite.expasy.org). The secondary structure of the protein was predicted using LYON-GERLAND PBIL (https://prabi.ibcp.fr); and the hydrophobic structure was predicted using ScanProsite (https: //prosite.expasy.org/scanprosite).

Expression Analysis by RT-PCR
For expression analysis in different tissues, roots, stems and leaves were harvested from 6-week-old alfalfa, while flowers were from 60-day-old plants. For the diurnal analysis, leaves were collected separately from 6-week-old alfalfa at zeitgeber time (ZT) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 time points, respectively. After extraction of total RNA, cDNA was prepared as described above. Semi-quantitative RT-PCR was carried out using gene-specific primers with MsActin serving as an internal control. The PCR product was analyzed in gel and images were captured using a Gel Image Analysis System FR-200A (Furi Science & Technology, Shanghai, China). For quantitative real-time PCR, SYBR Premix EX Taq TM qPCR Kit (Takara, Dalian, China) was used according to the instruction with BIO-RAD CFX96 TM Real-Time System (BioRad, Hercules, CA, USA). The Arabidopsis Actin gene was used as a loading control. The sequence of the primers used in this study is listed in Table S2.

Subcellular Localization Analysis
For the subcellular localization analysis of MsFTa-GFP fusion protein in onion epidermal cells, the construct of MsFTa-GFP was performed as follows: the MsFTa ORF sequence was subcloned into the pA7-GFP (35S::GFP) expression vector [29] using the primers of FT-GF and FT-GR containing XhoI and SpeI restriction sites, respectively. The plasmid of the pA7-GFP and 35S::MsFTa-GFP fusion binary vector was then transformed into separate onion epidermal cells via particle bombardment (GJ-1000, Scientz Biotechnology, Ningbo, China). The subcellular localization of the transiently expressed MsFTa-GFP fusion protein was imaged using a confocal laser-scanning microscope (Olympus FV500, Tokyo, Japan).

Plasmid construct and plant transformation
For generation of the transgenic Arabidopsis plants expressing 35S::MsFTa, first, a binary vector harboring 35S::MsFTa was constructed by amplifying MsFTa ORF with primers named pBI-FTa-f and pBI-FTa-r, and the sequence-confirmed MsFTa was introduced into the pBI121 vector (Clontech, Palo Alto, CA, USA) using Xba I and Sma I restriction sites. Secondly, the 35S::MsFTa plasmid was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis transformation was performed via the floral-dip method. T1 Arabidopsis seeds were screened on a MS medium containing 40 mg·L −1 kanamycin, and T2 lines confirmed by semi-quantitative RT-PCR were used in this study for MsFTa expression and phenotypic analysis. For Medicago transformation, explants from 4-week-old plants (R108 and fta1-1) growing under LDs were cocultivated with the agrobacterium containing the construct of 35S::MsFTa according to the Medicago handbook [45]. Kanamycin of 40 mg·L −1 was used to select transformant plants.

Conclusions
In this study, the FT homolog from alfalfa, one of the most important perennial forages, was isolated and characterized. MsFTa has highly identical sequence composition, especially exon/intron boundary and functional domains, with FTs in a variety of plant species. The diurnally regulated MsFTa is expressed preferentially in leaves and floral buds. Functionally, MsFTa accelerated flowering in Arabidopsis and the annual legume M. truncatula, indicating that the novel alfalfa FT acts as a flowering promoter.