Diversification in Functions and Expressions of Soybean FLOWERING LOCUS T Genes Fine-Tunes Seasonal Flowering

Abstract

The proper timing of flowering in response to environmental changes is critical for ensuring crop yields. FLOWERING LOCUS T (FT) homologs of the phosphatidylethanolamine-binding protein family play important roles as floral integrators in many crops. In soybean, we identified 17 genes of this family, and characterized biological functions in flowering for ten FT homologs. Overexpression of GmFT homologs in Arabidopsis revealed that a set of GmFT homologs, including GmFT2a/2b, GmFT3a/3b, and GmFT5a/5b, promoted flowering similar to FT; in contrast, GmFT1a/1b, GmFT4, and GmFT6 delayed flowering. Consistently, expressions of GmFT2a, GmFT2b, and GmFT5a were induced in soybean leaves in response to floral inductive short days, whereas expressions of GmFT1a and GmFT4 were induced in response to long days. Exon swapping analysis between floral activator GmFT2a and floral repressor GmFT4 revealed that the segment B region in the fourth exon is critical for their antagonistic functions. Finally, expression analysis of GmFT2a, GmFT5a, and GmFT4 in soybean accessions exhibiting various flowering times indicated that the mRNA levels of GmFT2a and GmFT5a were higher in early flowering accessions than in late-flowering accessions, while GmFT4 showed the opposite pattern. Moreover, the relative mRNA levels between GmFT2a/GmFT5a and GmFT4 was important in determining day length-dependent flowering in soybean accessions. Taken together, our results suggest that the functions of GmFT homologs have diversified into floral activators and floral repressors during soybean evolution, and the timing of flowering in response to changing day length is determined by modulating the activities of antagonistic GmFT homologs.

Keywords: FLOWERING LOCUS T; flowering time; functional diversification; photoperiods; soybean; soybean PEBP family.

FIGURE 1

Identification and sequence analysis of soybean PEBP family members. (A) Genomic organization of the soybean and Arabidopsis PEBP family members. Boxes and lines represent exonic and intronic regions, respectively. Numbers indicate the length of exons and introns (base pairs). The gene structures of soybean PEBP family members were determined on the basis of the alignment between the genomic and cDNA sequences. (B) Phylogenetic analysis of Arabidopsis and soybean PEBP family members. The phylogenetic tree was constructed using the Neighbor-Joining method of Mega 4 software program (Tamura et al., 2007) based on the amino acid sequences of the Arabidopsis and soybean PEBP family members. Bootstrap values (1,000 replicates) are indicated at the branches of the tree. (C) Partial amino acid sequence alignment of the 14-amino acid segment B region of soybean and Arabidopsis PEBP family members. Black stars above the upper row indicate the Tyr85(Y)/His88(H) and Gln140(Q)/Asp144(D) residues specifying Arabidopsis FT and TFL1 functions in flowering, respectively.

FIGURE 2

Expression analysis of soybean PEBP family members. Total RNAs were extracted from various tissues at different developmental stages of soybean plants grown in natural green house conditions. Transcript levels were analyzed by RT-PCR and subsequent Southern blotting. Soybean PBB2 (20S proteasome beta subunit) mRNA (Glyma14g01850) was used as a control (Thakare et al., 2010). Tl: trifoliate leaf (Tl1; oldest, Tl4; youngest), Ul: unifoliate leaf, Ep: epicotyl, Ct: cotyledon, Hy: hypocotyl, Rt: root, St: stem, Ab: axillary bud, Fl: flower. Seed weights of 50, 200, and 500 mg are weights of single seeds.

FIGURE 3

Effects of the ectopic expression of GmFT genes on flowering in transgenic Arabidopsis plants. (A) Phenotypes of 23-day old wild-type (Col-0) and transgenic Arabidopsis plants expressing soybean GmFT2a, GmFT2b, GmFT3a, GmFT3b, GmFT5a, and GmFT5b. (B) Phenotype of terminal flowers of 35S::GmFT2a-expressing Arabidopsis plants. Scale bar is 2 mm. (C) Phenotypes of 40-day old wild-type and transgenic Arabidopsis plants expressing GmFT1a, GmFT1b, GmFT4, and GmFT6. Wild-type and T1 transgenic plants were grown on the soil at 23°C under long-day conditions.

FIGURE 4

Diurnal expression of GmFT genes under LD and SD conditions. Total RNAs were extracted every 4 h from the first trifoliate leaves of 20-day old LD- and SD-grown plants, respectively. Relative mRNA levels of GmFT genes were analyzed by quantitative real-time PCR with three independent biological replicates and normalized to GmPBB2 mRNA. White and dark bars indicate light and dark phases, respectively. Data are shown as means ± standard deviation.

FIGURE 5

Flowering times of transgenic Arabidopsis plants expressing GmFT2a/GmFT4 exon swapping chimeras. The exons of GmFT2a and GmFT4 are shown as red and blue boxes, respectively. Segment B regions of GmFT2a and GmFT4 are highlighted by pink and cyan colors, respectively. The distribution of flowering times in LD conditions for T1 transformants and control plants (Col-0) are indicated by vertical bars; gray, red, dark blue, and light blue bars for Col-0, 35S::GmFT2a, 35S::GmFT4, and the chimeras, respectively. The number of plants is indicated above each bar.

FIGURE 6

Flowering phenotypes of transgenic Arabidopsis plants expressing the GmFT4 segment B-substitution mutants. (A) Amino acid sequences of the segment B regions of GmFT2a, GmFT4, and GmFT4 segment B-substitution mutants. The substituted amino acids of GmFT4 with corresponding amino acids of GmFT2a were indicated by red color. (B) Flowering times of GmFT4 segment B-substitution mutants. The distribution of flowering times in LD conditions for T1 transformants and control plants (Col-0) are indicated by vertical bars; gray, red, dark blue, and light blue bars for Col-0, 35S::GmFT2a, 35S::GmFT4, and the GmFT4 segment B-substitution mutants, respectively. The number of plants is indicated above each bar.

FIGURE 7

Expression of GmFT2a, GmFT5a, and GmFT4 genes in soybean accessions. (A) The number of days to flowering of 35 USDA soybean accessions grown in field conditions. (B) Evaluation of transcript levels of GmFT2a, GmFT5a, and GmFT4 in the third trifoliate (V3) leaves of 30-day old (V4 stage) plants by qRT-PCR with three independent biological replicates. Transcript levels were normalized to GmPBB2 mRNA levels. (C) Correlation analysis between expression levels of GmFT2a, GmFT5a, and GmFT4 mRNAs and flowering times of USDA soybean accessions. Data are shown as means ± standard deviation.

FIGURE 8

Expression of GmFT2a, GmFT5a, and GmFT4 mRNAs in leaves of early (Williams 82)- and late (PI229358)-flowering soybean accessions across different developmental stages. Fully expended trifoliate leaves from the top of the main stem were harvested from three independent plants grown in natural field conditions from 20 to 100 days after sowing. Relative mRNA levels of GmFT genes were analyzed by quantitative real-time PCR with three independent biological replicates and normalized to GmPBB2 mRNA. Days to flowering of Williams 82 (38.6D) and PI229358 (74.4D) are indicated by blue and red line, respectively. The result of independent RT-PCR experiments is also shown below each graph. Data is shown as mean ± standard deviation.