GmFT2a and GmFT5a redundantly and differentially regulate flowering through interaction with and upregulation of the bZIP transcription factor GmFDL19 in soybean

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Abstract

FLOWERING LOCUS T (FT) is the key flowering integrator in Arabidopsis (Arabidopsis thaliana), and its homologs encode florigens in many plant species regardless of their photoperiodic response. Two FT homologs, GmFT2a and GmFT5a, are involved in photoperiod-regulated flowering and coordinately control flowering in soybean. However, the molecular and genetic understanding of the roles played by GmFT2a and GmFT5a in photoperiod-regulated flowering in soybean is very limited. In this study, we demonstrated that GmFT2a and GmFT5a were able to promote early flowering in soybean by overexpressing these two genes in the soybean cultivar Williams 82 under noninductive long-day (LD) conditions. The soybean homologs of several floral identity genes, such as GmAP1, GmSOC1 and GmLFY, were significantly upregulated by GmFT2a and GmFT5a in a redundant and differential pattern. A bZIP transcription factor, GmFDL19, was identified as interacting with both GmFT2a and GmFT5a, and this interaction was confirmed by yeast two-hybridization and bimolecular fluorescence complementation (BiFC). The overexpression of GmFDL19 in soybean caused early flowering, and the transcription levels of the flowering identity genes were also upregulated by GmFDL19, as was consistent with the upregulation of GmFT2a and GmFT5a. The transcription of GmFDL19 was also induced by GmFT2a. The results of the electrophoretic mobility shift assay (EMSA) indicated that GmFDL19 was able to bind with the cis-elements in the promoter of GmAP1a. Taken together, our results suggest that GmFT2a and GmFT5a redundantly and differentially control photoperiod-regulated flowering in soybean through both physical interaction with and transcriptional upregulation of the bZIP transcription factor GmFDL19, thereby inducing the expression of floral identity genes. © 2014 Nan et al.

Figures

  • Figure 1. Overexpression of GmFT2a and GmFT5a causes precocious flowering in the soybean cultivar Williams 82. (A) The close shot of the transgenic plant in (B) shows the precocious flowers at the axils of the trifoliate leaves. (B) A transgenic GmFT2a plant showing precocious flowering at the axils of the trifoliate leaves. (C) A wild-type Williams 82 plant. (D) The close shot of the wild type Williams 82 plant in (C) does not show flowers at the axils of the trifoliate leaves. (E) The close shot of the transgenic plant in (F) shows the precocious flowers at the axils of the trifoliate leaves (F) A transgenic GmFT5a plant showing precocious flowering at the axils of the trifoliate leaves. (G) A wild-type Williams 82 plant. (H) The close shot of the wild-type Williams 82 plant in (G) does not show flowers at the axils of the trifoliate leaves. (I) Days to flowering from the emergence of the transgenic plants and wild-type plants. Averages and standard errors are calculated from four T2 plants for each construct and 5 Williams 82 plants. Double asterisks indicate significant differences from the corresponding wild-type Williams 82 at P,0.01. doi:10.1371/journal.pone.0097669.g001
  • Figure 2. Temporal and spatial expression of soybean flowering-related genes. (A) Transcript levels of eight soybean flowering-related genes (GmAP1a, GmAP1b, GmAP1c, GmAP1d, GmSOC1a, GmSOC1b, GmLFY1, GmLFY2) in leaves and shoot apices of the soybean cultivar Harosoy under SD (12L/12D) conditions; Tubulin is included as an endogenous control. Samples were collected from 10 DAE to 25 DAE. DAE: days after emergence. L: leaves; S: shoot apex. (B) Tissue-specific expression analyses of eight flowering-related genes by RT-PCR under SD (12L/12D) conditions. L: leaves, S: shoot apices, F: flowers, FB: flower buds, P: pods, R: roots. doi:10.1371/journal.pone.0097669.g002
  • Figure 3. GmFT2a and GmFT5a promote the expression of soybean flowering-related genes. (A) Expression analyses of GmFT2a, GmFT5a and flowering-related genes in transgenic GmFT2a plants (#2-1-1) and wild-type Williams 82 plants (WT). (B) Expression analyses of GmFT5a, GmFT2a and flowering-related genes in transgenic GmFT5a plants (#5-1) and wild-type Williams 82 plants (WT). The white and black columns represent relative expression in leaves and shoot apices, respectively. Asterisks and double asterisks indicate significant differences between transgenic and WT plants at 0.01,P,0.05 and P,0.01, respectively. doi:10.1371/journal.pone.0097669.g003
  • Figure 4. Seven GmFDLs (soybean FD-like genes) transcribed in leaves and shoot apices. (A) Transcript levels of seven GmFDLs in leaves and shoot apices of the soybean cultivar Harosoy under SD (12L/12D) conditions; Tubulin is included as an endogenous control. Samples were collected from 10 DAE to 25 DAE. L: leaves; S: shoot apices. (B) Multiple alignment of the amino acid sequences in the SAP motif region of the FDs from soybean and other species. The SAP motif is a putative sequence for FT binding. doi:10.1371/journal.pone.0097669.g004
  • Figure 5. Interactions of GmFDLs with GmFT2a and GmFT5a.
  • Figure 6. Overexpression of GmFDL19 in the soybean cultivar Dongnong 50 causes early flowering. (A) The close shot of the transgenic plant in (B) shows the precocious flowers at the axils of the trifoliate leaves (B) A transgenic GmFDL19 plant showing precocious flowering at the axils of the trifoliate leaves. (C) A wild-type Dongnong 50 plant. (D) The close shot of the wild-type Dongnong 50 plant in (C) does not show flowers at the axils of the trifoliate leaves. (E) Number of days to flowering in transgenic and wild-type plants. Averages and standard errors are calculated from five independent T2 plants and five Dongnong 50 plants. (F) Expression analyses of GmFDL19 and flowering-related genes in transgenic GmFDL19 plants (#12-1) and wild-type Dongnong 50 plants (WT); because these flowering related genes are transcribed mostly in shoot apices, shoot apex samples were collected from transgenic and wild-type Dongnong 50 plants at 40 DAE under LD (18L/6D) conditions. Asterisks and double asterisks indicate significant differences from the corresponding wild-type Dongnong 50 at 0.01,P,0.05 and P,0.01, respectively. doi:10.1371/journal.pone.0097669.g006
  • Figure 7. The GmFDL19 protein specifically binds with the ACGT core sequence in vitro. Potential bZIP binding sites presented in the GmAP1a promoter were used as probes in binding reactions with the purified recombinant GmFDL19 protein. The eight probes included seven potential bZIP binding sites and a CArG-box as negative control. (1) T-box (AACGTT), (2) A/C hybrid of A and C-box (TACGTC), (3) T/C hybrid of T and C-box (AACGTC), (4) CArG-box (CCNNNNNNNNGG), (5) G/A hybrid of G and A-box (CACGTA), (6) T/A hybrid of T and A-box (AACGTA), (7) G/A hybrid of G and A-box (CACGTA), (8) T-box (AACGTT). The scheme below indicates the positions of the various bZIP binding sites. doi:10.1371/journal.pone.0097669.g007
  • Figure 8. Proposed mechanism of photoperiod-regulated flowering controlled by GmFT2a and GmFT5a in soybean. (A) Model of GmFT2a and GmFT5a regulating the expression of GmAP1a. The horizontal dotted line represents the GmAP1a promoter, and the black vertical bars indicate the eight exons of GmAP1a. The green oval represents the GmFDL19 protein, and this protein can bind to the T-box, G-box or hybrid box (white rectangle) in the GmAP1a promoter. The orange oval represents the interactions of GmFT2a and GmFT5a with GmFDL19. (B) A proposed molecular network for photoperiod-regulated flowering in soybean. doi:10.1371/journal.pone.0097669.g008

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Nan, H., Cao, D., Zhang, D., Li, Y., Lu, S., Tang, L., … Kong, F. (2014). GmFT2a and GmFT5a redundantly and differentially regulate flowering through interaction with and upregulation of the bZIP transcription factor GmFDL19 in soybean. PLoS ONE, 9(5). https://doi.org/10.1371/journal.pone.0097669

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