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Identification and Molecular Characterization of <i>FKF1</i> and <i>GI</i> Homologous Genes in Soybean

<div><p>In <i>Arabidopsis</i>, FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) and GI (GIGANTEA) play important roles in flowering pathway through regulating daytime <i>CO</i> (<i>CONSTANS</i>) expression, and such a function is conserved across plants studied. But related reports are limited for soybean. In this study, we cloned <i>FKF1</i> and <i>GI</i> homologs in soybean, and named as <i>GmFKF1</i>, <i>GmFKF2</i>, <i>GmGI1</i>, <i>GmGI2</i>, and <i>GmGI3</i>, respectively. <i>GmGI1</i> had two alternative splicing forms, <i>GmGI1α</i> and <i>GmGI1β</i>. <i>GmFKF1</i>/<i>2</i> transcripts were diurnally regulated, with a peak at zeitgeber time 12 (ZT12) in long days and at ZT10 in short days. The diurnal phases between <i>GmGIs</i> transcript levels greatly differed. <i>GmGI2</i> expression was regulated by both the circadian clock and photoperiod. But the rhythmic phases of <i>GmGI1</i> and <i>GmGI3</i> expression levels were mainly conferred by long days. <i>GmFKFs</i> shared similar spatio-temporal expression profiles with <i>GmGIs</i> in all of the tissue/organs in different developmental stages in both LD and SD. Both GmFKF and GmGI proteins were targeted to the nucleus. Yeast two hybrid assays showed GmFKF1/GmFKF2 interacted with GmGI1/GmGI2/GmCDF1 (CYCLING DOF FACTOR CDF1 homolog in soybean); and the LOV (Light, Oxygen, or Voltage) domain in GmFKF1/GmFKF2 played an important role in these interactions. N-terminus of GmGI2 was sufficient to mediate its interaction with GmCDF1. Interestingly, N-terminus not full of GmGI3 interacted with GmFKF1/GmFKF2/GmCDF1. Ectopic over-expression of the <i>GmFKF1</i> or <i>GmFKF2</i> in <i>Arabidopsis</i> enhanced flowering in SD. Collectively, GmFKF and GmGI in soybean had conserved functional domains at DNA sequence level, but specific characters at function level with their homologs in other plants.</p></div

Identification of a Soybean <i>MOTHER OF FT AND TFL1</i> Homolog Involved in Regulation of Seed Germination

<div><p>Seed germination is an important event in the life cycle of seed plants, and is controlled by complex and coordinated genetic networks. Many genes involved in the regulation of this process have been identified in different plant species so far. Recent studies in both <i>Arabidopsis</i> and wheat have uncovered a new role of <i>MOTHER OF FT AND TFL1</i> (<i>MFT</i>) in seed germination. Here, we reported a homolog of <i>MFT</i> in soybean (<i>GmMFT</i>) which strongly expressed in seeds. Detailed expression analysis showed that the mRNA level of <i>GmMFT</i> increased with seed development but declined during seed germination. The transcription of <i>GmMFT</i> also responded to exogenous application of ABA and GA3. Ectopic expression of <i>GmMFT</i> CDS in <i>Arabidopsis</i> moderately inhibited seed germination. All these evidences suggest that <i>GmMFT</i> may be a negative regulator of seed germination.</p></div

Germination phenotype of <i>35S::GmMFT</i> transgenic <i>Arabidopsis</i> seeds.

<p>A, Germination phenotype of <i>35S::GmMFT</i> transgenic lines in Col background on 1/2 MS medium; B, Germination phenotype of <i>35S::GmMFT</i> transgenic lines in <i>mft-2</i> background on 1/2 MS medium; C, Germination phenotype of <i>35S::GmMFT</i> transgenic lines in <i>mft-2</i> background on 1/2 MS medium supplemented with 10 µM ABA. A significant difference in comparison with the “Col” for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099642#pone-0099642-g005" target="_blank">figure 5A</a> or the “<i>mft-2</i>” for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099642#pone-0099642-g005" target="_blank">figure 5B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099642#pone-0099642-g005" target="_blank">figure 5C</a> was indicated with an asterisk (P<0.05, Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test). Error bars denoted SEM.</p

Nuclear localization of GmFKF1, GmFKF2, GmGI1α, GmGI1β, GmGI2 and GmGI3 proteins in <i>Arabidopsis</i> protoplasts.

<p>The vector indicated the empty vector as negative control; The CFP was for CFP-AHL22, a nuclear marker (Xiao <i>et al</i>., 2009); the YFP was for YFP fluorescence; the red signal was due to auto-fluorescence of chloroplasts; the last panel showed superimposition of the former three panels.</p

Expression profiles of <i>GmGIs</i> during development.

<p><i>GmGI1α</i> (A), <i>GmGI1β</i> (B), <i>GmGI2</i> (C) and <i>GmGI3</i> (D) expression levels were performed in leaves. The samples and developmental stages in LD (left panel) and SD (right panel) were the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079036#pone-0079036-g006" target="_blank">Figure 6</a>. The soybean <i>UKN1</i> gene was used as the normalization transcripts.</p

Expression profiles of <i>GmGIs</i> in various tissues/organs.

<p>The expression of <i>GmGI1α</i> (A), <i>GmGI1β</i> (B), <i>GmGI2</i> (C) and <i>GmGI3</i> (D) were investigated in LD (left panel) and SD (right panel). The samples were collected as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079036#pone-0079036-g004" target="_blank">Figure 4</a>. The soybean <i>UKN1</i> gene was used as the reference gene.</p

The <i>FKF1</i> and <i>GI</i> ortholog genes in the soybean genome.

<p>(A) The gene structures of <i>GmFKF1</i> and <i>GmFKF2</i>, compared with that of <i>AtFKF1</i> (At1g68050.1). (B) The gene structures of <i>GmGI1α</i>, <i>GmGI1β</i>, <i>GmGI2</i>, and <i>GmGI3</i>, compared with that of <i>AtGI</i> (At1g22770.1). (C) A phylogenetic tree of the FKF1 proteins from soybean and other plant species. The protein sequences accessions used were AcFKF1 (<i>Allium cepa</i>, <i>GQ232754</i>), ZmFKF1 (<i>Zea mays</i>, <i>GRMZM2G107945</i>), ZmFKF2 (<i>Zea mays</i>, <i>GRMZM2G106363</i>), SbFKF1 (<i>Sorghum bicolor</i>, <i>Sb05g021030</i>), OsFKF1 (<i>Oryza sativa</i>, <i>Os11g34460</i>), BdFKF1 (<i>Brachypodium distachyon</i>, <i>Bradi4g16630</i>), TaFKF1(<i>Triticum aestivum</i>, <i>ABL11478.1</i>), HaFKF1 (<i>Helianthus annuus</i>, <i>ADO61006.1</i>), AtFKF1 (<i>Arabidopsis thaliana</i>, <i>At1g68050.1</i>), MtFKF1 (<i>Medicago truncatula</i>, <i>Medtr4g156890</i>), GmFKF1 (<i>Glycine max</i>, <i>Glyma05g34530</i>), GmFKF2 (<i>Glycine max</i>, <i>Glyma08g05130</i>), McFKF1 (<i>Mesembryanthemum crystallinum</i>, <i>AAQ73528.1</i>), HvFKF1 (<i>Hordeum vulgare</i>, <i>ACR15149.1</i>). (D) The phylogenetic relationships among GI homologs. Accession numbers: AcGI (<i>Allium cepa</i>, <i>GQ232756</i>), OsGI (<i>Oryza sativa</i>, <i>Os01g0182600</i>), SbGI (<i>Sorghum bicolor</i>, <i>Sb03g003650</i>), ZmGI (<i>Zea mays</i>, <i>ABZ81992.1</i>), ZmGI1A (<i>Zea mays</i>, <i>DAA06172.1</i>), LpGI (<i>Lolium perenne</i>, <i>CAY26028.1</i>), TaGI1 (<i>Triticum aestivum</i>, <i>AAQ11738.1</i>), TaGI2 (<i>Triticum aestivum</i>, <i>AAT79486.1</i>), TaGI3 (<i>Triticum aestivum</i>, <i>AAT79487.1)</i>, HvGI (<i>Hordeum vulgare</i>, <i>AAW66945.1</i>), ScGI (<i>Secale cereal</i>, <i>ADR51711.1</i>), BdGI (<i>Brachypodium distachyon</i>, <i>DV476579</i>), LgGI (<i>Lemna gibba</i>, <i>BAD97869.1</i>), MtGI (<i>Medicago truncatula</i>, <i>XP_003592048.1</i>), PsGI (<i>Pisum sativum</i>, <i>ABP81863.1</i>), GmGI1α (<i>Glycine max</i>, <i>Glyma20g30980</i>), GmGI1β (<i>Glycine max</i>, <i>Glyma20g30980</i>), GmGI2 (<i>Glycine max</i>, <i>Glyma09g07240</i>), GmGI3 (<i>Glycine max</i>, <i>Glyma10g36600</i>), PtGI (<i>Populus trichocarpa</i>, <i>XP_002300901.1</i>), AtGI (<i>Arabidopsis thaliana</i>, <i>At1g22770.1</i>). (C) and (D) phylogenetic trees were constructed with MEGA 4.0 software. Full-length amino acid sequences were aligned and Bootstrap analysis was performed based on 1,000 replicates.</p

Interactions between GmFKFs and GmGI in yeast.

<p>(A) GmFKFs interacted with GmGIs and GmCDF1. GmFKFs (LF) included the LOV domain and F-box motif of GmFKF1 and GmFKF2. (B) GmGIs N interacted with GmFKFs (LF) and GmCDF1. GmGIs N represented the N terminus of GmGIs. -LW, synthetic dropout (SD) yeast growth medium lacking leucine and tryptophan; -LWHA, SD medium lacking Leu, Trp, histidine, and adenine.</p

Circadian rhythms of <i>FKF1</i> homologs in soybean.

<p><i>GmFKF1</i> (A) and <i>GmFKF2</i> (B) gene expression under different light regimes. LD, 16 hr light/8 hr dark; SD, 8 hr light/16 hr dark; LL, constant light; DD, constant dark. Soybean gene <i>ACT11</i> was used as a control for normalization.</p