Identification and Characterization of FaFT 1 : A Homolog of FLOWERING LOCUS T from Strawberry

FLOWERING LOCUS T (FT)-like genes play crucial roles in flowering transition in several plant species. In this study, a homolog of FT, designated as FaFT1, was isolated and characterized from strawberry. The open reading frame of FaFT1 was 531 bp, encoding a protein of 176 amino acids. Phylogenetic and sequence analysis showed that the FaFT1 protein contained the conservation of Tyr84 and Gln139, as well as the highly conserved amino acid sequences LGRQTVYAPGWRQN and LYN and that it was a member of the FT-like genes of dicots. Subcellular localization analysis revealed that the FaFT1 protein mainly localized in the nuclei of the Arabidopsis protoplasts. FaFT1 was highly expressed in strawberry mature leaves and its expression level decreased under floral induction conditions. Additionally, FaFT1 expression exhibited diurnal circadian rhythm both under SD and LD conditions. Over expression of FaFT1 in wild-type Arabidopsis caused early flowering. Taken together, these results indicate that FaFT1 is a putative FT homolog in strawberry, acting as a floral promoter in Arabidopsis.


INTRODUCTION
The transition from vegetative into reproductive phase is a crucial developmental process of flowering plants.The genetic network of flowering has been most extensively studied in the annual model plant Arabidopsis thaliana and six genetically pathways have been identified that control flowering: the vernalization, photoperiod, autonomous, ambient temperature, gibberellin and age pathways (Fornara et al., 2010).These signaling pathways converge to regulate a small number of floral integrator genes, which include FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), LEAFY (LFY) and FLOWERING LOCUS C (FLC) (Boss et al., 2004;Parcy, 2005).Among these genes, FT plays a central role in integrating flowering signals from the photoperiodic, vernalization and autonomous pathways (Boss et al., 2004).
The expression of FT is directly regulated by the CONSTANS (CO) and GIGANTEA under inductive photoperiod (Suárez-López et al., 2001;Sawa and Kay, 2011).CO is directly binds to the FT promoter or indirectly acts with other transcriptional factors to regulate FT expression (Wenkel et al., 2006;Kobayashi and Weigel, 2007).The MADS-box proteins FLC and SHORT VEGETATIVE PHASE (SVP) form a complex that inhibits FT expression by binding to the CArG box of FT genomic sequence (Li et al., 2008).The SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) can also regulate FT expression to control ambient temperature-responsive flowering (Kim et al., 2012).Furthermore, FT expression is also affected by six miR172-targeted AP2-like genes and two TEMPRANILLO proteins (TEM1and TEM2) (Castillejo and Pelaz, 2008;Mathieu et al., 2009).
The ectopic expression of FT orthologs in Arabidopsis and the other plant species have shown that FT-like proteins act as universal flowering promoters (Pin and Nilsson, 2012).Although FT-like genes play conserved roles in promoting flowering, their expression pattern differs.For example, the Malus MdFT1 is mainly expressed in the apical meristem during the floral transition, whereas expression of MdFT2 peaks at the reproductive tissues and developing fruits (Kotoda et al., 2010).In Populus, the PtFT1 gene is preponderantly expressed in late winter, PtFT2 is expressed in the vegetative growth period (Hsu et al., 2011).
Strawberries (Fragaria spp.) are members of perennial rosette plants, belonging to the most economically significant berry crops.Cultivated strawberry (Fragaria×ananassa Duch.) is a kind of the most popular soft fruit species because of its unique flavor and nutrient values.The proper time of flowering has significant influences on yield, fruit quality and shelf life of strawberry.Most varieties of cultivated strawberry and wild strawberry (Fragaria vesca L.) are temperature-dependent Short Day (SD) plants, which flower under decreased photoperiod conditions in autumn (Heide and Sønsteby, 2007).Therefore, the photoperiod plays a key role in the flowering time control of strawberry.In 2012, Koskela et al. (2012) used a wild diploid strawberry, Fragaria vesca, to elucidate the photoperiodic flowering in strawberry.The expression and function FvFT1 were studied in F. vesca.However, there were also some other questions need to be study further, which included the ectopic expression in Arabidopsis and subcellular localization analysis.Recent studies have shown that there are some differences in SOC1-like gene expression between wild diploid and cultivated octoploid strawberry (Lei et al., 2013;Mouhu et al., 2013).
In this context, a FT-like gene, FaFT1, was isolated and characterized from cultivated strawberry cv.Camarosa.The sequence information, subcellular localization and expression pattern of FaFT1 were investigated.We also analyzed the function of FaFT1 in flowering time using transgenic Arabidopsis.

MATERIALS AND METHODS
Plant material: Cultivated strawberry (Fragaria× ananassa Duch.cv.Camarosa) runner plants with 3-4 leaves were used for the experiments.The plants were kept in a greenhouse maintained at 22°C under long day (LD) conditions (16 h light/8 h dark) with high pressure sodium lamps (~235 µmol/m 2 /s) to prevent floral induction.To monitor changes in gene expression in the leaves during floral induction, the plants were transferred to SD conditions (8 h light/16 h dark) with temperature maintained at 18/15°C (day/night) for six weeks for floral induction, pieces of young fully developed leaves were collected at seven different time points with one-week intervals.Samples of root, stem, leaf, flower bud, flower, stamen, pistil, sepal, petal, receptacle and fruit were collected for tissue-specific expression assays.In diurnal expression analyses, pieces of young fully developed mature leaves were sampled every 2 h after dawn for 24 h as a bulk of 10 plants grown under SD or LD conditions.
Arabidopsis thaliana Columbia-0 (Col-0) was used as the wild-type.The wild-type or transgenic plants were grown at 22±2°C under LD conditions with white fluorescent lights (~150 µmol/m 2 /s).For analysis of the flowering time phenotype, plants were grown in soil under LD conditions, flowering time was determined by counting the total number of rosette leaves at the time of floral bolting.

RNA preparation and cDNA cloning:
Total RNA was extracted by the pine tree method (Chang et al., 1993) and the first-strand cDNA was constructed by oligo (dT15) primer with M-MLV reverse transcriptase (Promega, Madison, USA) according to the manufacturer's instructions.To clone the FaFT1 gene, the FT protein (GenBank ID: Q9SXZ2) was used for a BLAST search in the strawberry genome GBrowse (http://www.strawberrygenome.org/) and a high homology protein with the gene locus 21535 was found.Then the specific primers for full-length of cDNA cloning were designed for FaFT1 (forward, 5′-CAG CTA GCT AGC TTG AAG GAT C-3′; reverse, 5′-GCG AAT TAT GAC ATG CAT GTA CT-3′) using the Primer Premier 5.0 program.PCR was performed with Pfu DNA polymerase (Promega, Madison, USA) under the following conditions: 95°C for 1 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s and 72°C for 1 min.The amplified PCR fragments were cloned into pSIMPLE-19 EcoRV/BAP vector (Takara, Dalian Division, China) and sequenced.

Subcellular localization assay:
The open reading frame (ORF) of FaFT1 was amplified with the primer pair: 5′-AAG CTT CAT GCC TAG GGA CAG G-3′ (HindIII site underlined) and 5′-GGT ACC GCG ATG ATC TTC TCC T-3′ (KpnI site underlined).The PCR product was cloned into the pE3025-GFP vector to generate pE3025-FaFT1-GFP.For protoplast transient expression assay, the mesophyll protoplasts were isolated from the rosette leaves of four-week-old plants of wild-type Arabidopsis.The transformation of protoplasts was performed as described by Yoo et al. (2007).The transformed Arabidopsis protoplasts were incubated for 16 h at 22°C in darkness and GFP fluorescence was observed with a NIKON ECLIPSE TE2000-E confocal laser-scanning microscope (Nikon, Tokyo, Japan).

Gene expression assay:
Total RNA isolation and cDNA synthesis were performed as described above.The cDNA samples were diluted 1:10 with water, 2 µL of the diluted cDNA was used as a template for quantitative real-time PCR (qRT-PCR) reaction.The qRT-PCR analysis was performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster, USA) using UltraSYBR Mixture (CWBIO, Beijing, China).Reactions were performed by an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 10 s at 95°C and 31 s at 60°C.Each sample represented three biological replicates, each of them included four technical replicates.Data was analyzed using the 2 −∆∆CT method as described by Livak and Schmittgen (2001).The primers used in this qRT-PCR were listed below: 5′-CAG ACC AGC AGA GGC TTA TCT T-3′ (forward) and 5′-TTC TGG ATA TTG TAG TCT GCT AGG G-3′ (reverse) for FaUBI (Mouhu et al., 2009); 5′-GAA GAG TCA TAG GTG ATG TTC TGG A-3′ (forward) and 5′-ACC ATT GTT GAC CTC CTT AGA AGT-3′ (reverse) for FaFT1.The FaUBI was used as the housekeeping gene.

Plant transformation:
The protein-coding region of FaFT1 cDNA was amplified using the primer pair: 5′-GGA TCC ATG CCT AGG GAC AGG-3′ (BamHI site underlined) and 5′-TCT AGA TTA CGA TGA TCT TCT CCT TCC-3′ (XbaI site underlined).The PCR product was ligated into the binary plant transformation vector pCBI302-3 under the control of the Cauliflower mosaic virus (CaMV) 35S promoter.The plasmid was transformed into Agrobacterium tumefaciens strain GV3101 and then transformed into wild-type Arabidopsis plants using the floral dip method (Clough and Bent, 1998).Transgenic seedlings were selected by spraying BASTA (Bayer CropScience, Wolfenbüttel, Germany).Two independent lines of the T 2 generation were randomly chosen for morphological analysis.

RESULTS AND DISCUSSION
Cloning and sequence analysis of FaFT1: The ORF of the FaFT1 gene was 531 bp (GenBank ID: KP184716), encoding a protein of 176 amino acids with an estimated molecular mass of 19.7 kDa and an isoelectric point of 8.3.The FaFT1 protein showed 92, 92, 91, 90 and 89% identity to FTs from Prunus mume, Populus tomentosa, Malus domestica, Gossypium hirsutum and Betula platyphylla, respectively.The FaFT1 protein had the conserved key amino acid residue Tyr84 (Y) and Gln139 (Q) in the positions corresponding to Tyr85 and Gln140 of Arabidopsis FT (Fig. 1).Tyr85/His88 and Gln140/Asp144 are likely to be responsible for the differences in function between FT and TFL1 (Hanzawa et al., 2005;Ahn et al., 2006).FaFT1 also contained two highly similar sequences to Arabidopsis FT in the 14-amino acid stretch referred to as "segment B" and in the LYN triad in "segment C" (Ahn et al., 2006) (Fig. 1).To further evaluate the relationship between members of the FT homologous genes, a phylogenetic tree was constructed using their amino acid sequences.The reconstructed tree indicated that the FT-like gene family could be separated into dicot and monocot clades, with FaFT1 protein belonging to the dicot clade together with other FT homologs from the Rosaceae (Fig. 2), suggesting that FaFT1 was a putative ortholog of FT in strawberry.
FaFT1 is targeted to the nucleus: For the subcellular localization assay, the coding region of FaFT1 was fused to GFP to form the fusion gene FaFT1-GFP driven by the CaMV 35S promoter in pE3025.The fusion gene plasmid and GFP control plasmid were transformed into Arabidopsis mesophyll protoplasts.The fluorescence of FaFT1-GFP was observed exclusively in the nuclei of the Arabidopsis protoplasts, whereas, as expected, the GFP protein was detected throughout the cells (Fig. 3).Thus, FaFT1 appeared to be a nucleus-localized protein.This result is consistent with the SFT (SINGLE-FLOWER TRUSS) protein of tomato (Lifschitz et al., 2006), but different from MdFT2 of apple (Li et al., 2010), which localizes in the membrane.Our results suggested that FaFT1 might act as a transcriptional factor in the nucleus.

Expression analysis of the FaFT1 gene:
To characterize the expression pattern of FaFT1, we first examined the tissue-specific expression of FaFT1 by qRT-PCR using various tissues.FaFT1 transcripts were almost undetectable in fruits and roots (Fig. 4), whereas its expression in mature leaves, floral buds, flowers and shoot apices significantly increased, with the highest level in mature leaves.This observation indicated that FaFT1 might be closely associated with photoperiodic response for flowering and reproductive development in strawberry.Although FT is a mobile signal originating from leaves (Corbesier et al., 2007;Tamaki et al., 2007), many FT homologs are highly expressed in reproductive tissues, such as flower buds and flowers in Jatropha curcas (Li et al., 2014), flower buds in F. vesca (Koskela et al., 2012), young ovary of mature flowers in Cymbidium goeringii (Xiang et al., 2012).Actually, FT and its homologs play multiple roles in plant development in addition to flowering time control (Pin and Nilsson, 2012).
To detect the involvement of FaFT1 in floral transition of strawberry, we monitored the change of FaFT1 expression in leaves of strawberry during the floral induction.For the experiment, strawberry runner plants were grown under SD conditions for six weeks for floral induction.Interestingly, in our study, FaFT1 expression level decreased under inductive SDs (Fig. 5).This result showed that the FaFT1 transcripts were negatively correlative with photoperiod requirement for flowering and contrasted to the situation of GmFT2a, GmFT5a and CsFTL3 in other SD plants (Kong et al., 2010;Oda et al., 2012).
Although the FaFT1 expression pattern under SDs was similar to that of FvFT1 in F. vesca (Koskela et al., 2012), the FaFT1 expression decreased gradually in a mild manner during the floral induction.
The diurnal circadian rhythm of gene expression was then analyzed by qRT-PCR for FaFT1 in leaves under SD and LD conditions.As shown in Fig. 6, FaFT1 exhibited a diurnal circadian rhythm both under SD and LD conditions.Under LDs, FaFT1 transcripts increased during the dark phase of the day, peaking around the middle of the dark phase.Interestingly, FaFT1 also exhibited a trimodal expression pattern in SDs, with peak expression at 3 h before and after dawn, 1 h after dusk (Fig. 6).Although expression level was very low under SDs, FaFT1 showed a diurnal circadian rhythm.This result was different from the FvFT1 expression in diploid F. vesca, in which the diurnal rhythm of FvFT1 expression is present only under LDs (Koskela et al., 2012).Taken together, our results revealed that there might be complex regulatory mechanisms to control FaFT1 expression in the cultivated octoploid strawberry.

CONCLUSION
In summary, a homolog of FT, designated as FaFT1, was isolated and characterized from strawberry.FaFT1 had high identity to other FT homologs and contained the highly conserved amino acid sequences LGRQTVYAPGWRQN and LYN.FaFT1 protein mainly localized in the nuclei of the Arabidopsis protoplasts.FaFT1 was highly expressed in strawberry mature leaves and its expression level decreased under floral induction conditions.Additionally, FaFT1 expression exhibited diurnal circadian rhythm both under SD and LD conditions.FaFT1 acted in Arabidopsis as a flowering activator and played a conserved role in regulating floral transition.

Fig. 1 :
Fig.1: Amino acid sequences alignment of FaFT1 and its homologs from other plant species.The proteins were initially aligned using Clustal W and edited with BioEdit.Identical and similar amino acids were presented by black and gray shading, respectively.The conserved residues Tyr and Gln were indicated, the 14-amino-acid stretch (segment B) and the LYN triad (segment C) were marked with double line

Fig. 5 :
Fig. 5: Expression pattern of FaFT1 in the shoot apex under floral induction conditions.Each column represented the mean of three replicates.Error bars on each column represented the standard error (S.E.) of three replicates

Table 1 :
Number of days to flowering and the number of rosette leaves at flowering of wild-type and 35S::FaFT1 Arabidopsis under LD

expression of FaFT1 promotes flowering in Arabidopsis:
To investigate the function of FaFT1, transgenic Arabidopsis plants that over expressed FaFT1 via the CaMV 35S promoter were generated and two independent T 2 lines were selected for flowering time analysis under LD conditions.The transgenic lines flowered earlier than wild-type plants under LD conditions (Fig.7): Transgenics started bolting with 9.38 or 10.19 rosette leaves compared to 12.73 rosette leaves for wild-type plants.As shown in Table1, Arabidopsis wild-type plants required approximately 30 d to flowering, whereas the transgenic lines only required 24~25 d to flowering.In addition, the transgenic lines showed no obviously morphological changes.These results indicated that FaFT1 acted in Arabidopsis as a flowering activator, promoting the transition from the vegetative phase to the reproductive phase.Further study on the floral-related genes expression are needed to elucidate the mechanism of FaFT1 that involved in accelerating flowering of transgenic Arabidopsis.