<i>GmFT4,</i> a Homolog of <i>FLOWERING LOCUS T</i>, Is Positively Regulated by <i>E1</i> and Functions as a Flowering Repressor in Soybean

<div><p>The major maturity gene <i>E1</i> has the most prominent effect on flowering time and photoperiod sensitivity of soybean, but the pathway mediated by <i>E1</i> is largely unknown. Here, we found the expression of <i>GmFT4</i>, a homolog of <i>Flowering Locus T,</i> was strongly up-regulated in transgenic soybean overexpressing <i>E1</i>, whereas expression of flowering activators, <i>GmFT2a</i> and <i>GmFT5a</i>, was suppressed. <i>GmFT4</i> expression was strongly up-regulated by long days exhibiting a diurnal rhythm, but down-regulated by short days. Notably, the basal expression level of <i>GmFT4</i> was elevated when transferred to continous light, whereas repressed when transferred to continuous dark. <i>GmFT4</i> was primarily expressed in fully expanded leaves. Transcript abundance of <i>GmFT4</i> was significantly correlated with that of functional <i>E1,</i> as well as flowering time phenotype in different cultivars. Overexpression of <i>GmFT4</i> delayed the flowering time in transgenic <i>Arabidopsis</i>. Taken together, we propose that <i>GmFT4</i> acts downstream of <i>E1</i> and functions as a flowering repressor, and the balance of two antagonistic factors (<i>GmFT4</i> vs <i>GmFT2a</i>/<i>5a</i>) determines the flowering time of soybean.</p></div

Expression analysis of <i>FT</i>-like genes in transgenic soybean overexpressing <i>E1</i> and WT plants under LDs.

<p>Fully expanded trifoliolate leaves were sampled 4-quantitative RT-PCR. SOV#L1, SOV#L2 and SOV#L3 were T₂ transgenic plants from transgenic T₀ line TG4, that has three copy exogenous <i>E1</i> insertions. SOV#L4 was T₂ transgenic plant from transgenic T₀ line TG2, that has 7–8 copy exogenous <i>E1</i> insertions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089030#pone.0089030-Xia1" target="_blank">[37]</a>. SVC, transformation vector only (i.e., vector control); WT, Kariyutaka. The <i>TUA5</i> gene was used as a control.</p

Expression analysis of <i>GmFT4</i> in different soybean cultivars under SDs and LDs by real-time RT-PCR.

<p>(<b>A</b>) Number of days to flowering. (<b>B</b>) Evaluation of <i>GmFT4</i> transcript levels in fully expanded trifoliolate leaves by real-time RT-PCR. Transcript levels relative to <i>TUA5</i> were represented in each treatment; Soybean cultivar Kariyutaka under SDs was used as control. Values represent means of three biological replicates; error bars indicate standard deviation.(<b>C and D</b>) Correlation analysis between <i>GmFT4</i> mRNA and flowering time of different soybean cultivars under LDs and SDs. Results showed that <i>GmFT4</i> mRNA expression was significantly correlated with flowering time of different soybean cultivars under both SDs and LDs. (<b>E and F</b>) Correlation analysis between <i>GmFT4</i> mRNA expression and <i>E1</i> mRNA expression in cultivars carrying <i>E1</i> allele and cultivars carrying <i>e1-as</i> allele. <i>GmFT4</i> expression is significantly correlated with the <i>E1</i> expression in cultivars carrying both <i>E1</i> allele and cultivars carrying <i>e1</i>-as allele.</p

<i>GmFT4</i> expression is associated with flowering time, <i>E1</i> genotype and <i>E1</i> expression.

<p>Plants were grown in a climate chamber under either SDs (12 h:12 h light/dark) or LDs (16∶8 h light/dark). Fully expanded trifoliolate leaves were sampled 4 h after beginning of light phase from three individual plants. Relative expression level of <i>GmFT4</i> and <i>E1</i> were analyzed by real-time RT-PCR. Transcript levels relative to <i>TUA5</i> are represented in each treatment, s.d. represents standard deviation. Soybean cultivar Kariyutaka under SDs was used as control whose expression level was set to 1 for all genes analyzed. Values represent means of three biological replicates. Genotype <i>E1</i> is considered as functional WT allele, the <i>e1-as</i> allele represents a partially functional allele and the <i>e1-fs</i> alleles are nonfunctional allele.</p

Tissue-organ expression analysis of <i>GmFT4</i> in different soybean cultivars.

<p>All tissues were sampled 2-time RT-PCR analysis. Transcript levels relative to <i>TUA5</i> are represented in each treatment. Values represent means of three biological replicates; error bars indicate standard deviation. (<b>A</b>) Tissue-specific organ expression analysis of <i>GmFT4</i> in soybean cultivars Kariyutaka, Harosoy-<i>E1</i>, Harosoy-<i>e1</i> and HX3. (<b>B</b>) Tissue-organ expression analysis of <i>E1</i> in <i>E1</i> overexpressing transgenic soybean and WT (Kariyutaka). (<b>C</b>) Tissue-organ expression analysis of <i>GmFT4</i> in <i>E1</i> overexpressing transgenic soybean and WT (Kariyutaka).</p

Sequence comparison of FT/TFL1 family members from flowering plants.

<p>(<b>A</b>) Alignment of sequences of FT/TFL1 family members from flowering plants. The Tyr85/His88 residue, that lies at the entrance to the ligand-binding pocket, distinguishing all FT from TFL1 members is boxed in red. Segment B is boxed in black: the Asp144/Gln140 residue distinguishing all FT from TFL1 members is indicated by red arrow. The predicted key residue, which may play an important role in functional diversification is indicated by green arrow. (<b>B</b>) Phylogenetic tree of GmFT4 and other FT/TFL1 family members, most of which have been functionally characterized.</p

The distribution of flowering time (R1) of F₂ populations derived from Kariyutaka × Moshidougong 503 at Harbin in 2013 (A), in 2014 (B and C), and at Hailun in 2014 (D).

<p>The flowering time (R1) for parents are indicated by arrows; DAE, days after emergence.</p

The distribution of flowering time (R1) of an F₂ population derived from Kariyutaka × Suzumaru at Harbin in 2013 (A) and in 2014 (B). The flowering time (R1) for parents are indicated by arrows; DAE, days after emergence.

<p>The distribution of flowering time (R1) of an F₂ population derived from Kariyutaka × Suzumaru at Harbin in 2013 (A) and in 2014 (B). The flowering time (R1) for parents are indicated by arrows; DAE, days after emergence.</p

Alignment of <i>e1-ba3</i> with other known <i>E1</i> mutations.

<p>The SNP mutation site for <i>e1-as</i>, <i>e1-fs</i> are marked by black boxes; and the 5 bp mutation is marked with a red box. The bipartite nuclear localization sites are marked by black dashed line. The B3-like domain is also indicated with a dashed red line.</p

Diurnal Expression Pattern, Allelic Variation, and Association Analysis Reveal Functional Features of the <i>E1</i> Gene in Control of Photoperiodic Flowering in Soybean

<div><p>Although four maturity genes, <i>E1</i> to <i>E4</i>, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the <i>E1</i> gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the <i>E1</i> gene is associated with flowering time phenotype. The bimodal expression of the <i>E1</i> gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of <i>E1</i> was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an <i>e1-b3a</i>, which occurs in the middle of B3 domain of the <i>E1</i> gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the <i>E1</i> locus significantly underlay flowering time in three F₂ populations. Taken together, we can conclude the legume specific <i>E1</i> gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the <i>E1</i> gene will extend our understanding of the soybean flowering pathway in soybean.</p></div