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

Abstract

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 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.

Keywords: MsFTa; alfalfa; flowering time; transgenic plants.

Figure 1

Phylogenetic analysis of MsFTa and its orthologs in the indicated plant species. The full-length sequence of 33 FT and FT-like proteins from six model species was analyzed using DNAMAN (version 7.0). The numbers at the nodes of the neighbor-joining tree indicate the bootstrapping values based on 1000 interactions. MsFTa was underlined and Mt6g033440, a novel FT in M. truncatula was boxed. Gene accession numbers: MsFTa (GenBank: JF681135); Arabidopsis thaliana (At): AtFT (At1g65480); Medicago truncatula (Mt): MtFTa1 (Mt7g084970), MtFTa2 (Mt7g085020), MtFTb1 (Mt7g006630), MtFTb2 (Mt7g006690), MtFTc (Mt7g085040), MtFT (Mt6g033040); 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.

Figure 2

Sequence alignment of FTs in the indicated species. Sequence was aligned using DNAMAN Version 7 (Lynnon Corporation, Quebec, Canada). Homology level was highlighted by shading in color: black for 100%, pink for ≥75%, blue for ≥50% and yellow for ≥33% identity. The ligand-binding motif and the external loop of 14-amino acid stretch were underlined. The amino acid residues encoded by exon 2 (62 bp) and exon 3 (41 bp) were underlined with a dashed line. Triangles indicated the conserved amino acids in the ligand-binding pocket. An asterisk (*) indicated the conserved key residue Tyrosine (Y) at position 85 of MsFTa.

Figure 3

Schematic representation of the exon/intron structure of FTs in the indicated species. The information of FT gene composition is from EnsemblPlants (http://plants.ensembl.org). Gene structure was drawn using DNAMAN (Version 7). Solid lines represent introns and black boxes represent the exons with length indicated.

Figure 4

Subcellular localization of the MsFTa-GFP fusion protein in onion epidermal cells. The MsFTa-GFP fused construct and control vector pA7-GFP [29] were transiently transformed into onion epidermal cells by microprojectile bombardment. All images were captured using a confocal laser scanning microscope (Olympus FV500). (a–c): Image of onion epidermal cells expressing 35S::MsFTa-GFP taken under GFP fluorescence (b) or in bright field (c), the merged image was shown in (a); (d–f): Image of onion epidermal cells expressing 35S::GFP taken under GFP fluorescence (e) or in bright field (f), the merged one was shown in (d). Bar = 100 µm.

Figure 5

Diurnal and tissue-specific expression of MsFTa. (a) The expression level of MsFTa transcript during 24 h zeitgeber time (ZT) under LD. The white bar represents day time and the black bar represents night. (b) Relative expression of MsFTa in roots, stems, leaves and flowers of alfalfa at ZT 14. Bars represent the means ± SD of three biological replicates.

Figure 6

Ectopic expression of MsFTa in Arabidopsis caused early flowering. (a) Verification of two independent transgenic Arabidopsis lines (T2-1 and T2-4 of the T2 generation) harboring 35S::MsFTa construct by PCR. (b) Transcriptional analysis of MsFTa in the two transgenic lines verified in (a). Arabidopsis Actin 2 (AT3g18780) was used as internal control. Leaves from two-week old plants were harvested for total RNA extraction. (c) Three-week-old plants of Col-0 and MsFTa over-expressing line (T2-4). (d,e) Flowering time analysis of the over-expression T2 plants grown under LD conditions in terms of days after germination (d) and the No. of rosette leaves (e). At least 15 plants from three batches were scored for each line. The significant difference between the two genotypes was indicated as * (Student’s t-test, p < 0.05). (f) Four-week-old plants of Col-0, ft-10 and the transgenic ft-10 expressing 35S::MsFTa (comp), showing rescued flowering time. (g) Statistical analysis of the No. of rosette leaves upon the emergence of the first flower. * indicated the significant difference from wild type (Student’s t-test, p < 0.05).

Figure 7

Ectopic expression of MsFTa in M. truncatula caused early flowering. (a) Comparison of flowering time between Medicago cv R108 overexpressing 35S::MsFTa (T1) and control (R108) plants under LD conditions. (b) Flowering time analysis of M. truncatula fta1-1 mutant (NF3307) plants ectopically expressing 35S::MsFTa (T1) 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 from wild type (Student’s t-test, p < 0.05).