Identification of a soybean MOTHER OF FT AND TFL1 homolog involved in regulation of seed germination

Abstract

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 Arabidopsis and wheat have uncovered a new role of MOTHER OF FT AND TFL1 (MFT) in seed germination. Here, we reported a homolog of MFT in soybean (GmMFT) which strongly expressed in seeds. Detailed expression analysis showed that the mRNA level of GmMFT increased with seed development but declined during seed germination. The transcription of GmMFT also responded to exogenous application of ABA and GA3. Ectopic expression of GmMFT CDS in Arabidopsis moderately inhibited seed germination. All these evidences suggest that GmMFT may be a negative regulator of seed germination.

Figure 1. Bioinformatics analysis of MFT-like proteins.

A, Alignment of amino acid sequences of MFT-like proteins in soybean, wheat, Arabidopsis. The black box indicates N-terminus extension of GmMFT; White letters in black background indicate the identical amino acid residues between only soybean and wheat; Black arrow indicates His/Tyr residue involved in FT/TFL1 function switching; Dotted arrows indicate conserved D-P-D-x-P and G-x-H-R motifs of MFT proteins. B, Phylogenetic analysis of MFT-like proteins. A neighbor-joining tree was constructed with MEGA 4.0 software based on the amino acid sequences. Bootstrap values were provided to show reliability at each node. The sequences are from Zea mays (ZCN9, ABX11011; ZCN10, ABW96233; ZCN11, ABX11013), Oryza sativa (OsMFT1, Os06g30370; OsMFT2, Os01g02120), Triticum aestivum (TaMFT, BAK78908), Hordeum vulgare (HvMFT, BAH24198), Arabidopsis (AtMFT, At1g18100), Glycine max (GmMFT, Glyma05g34030), Medicago truncatula (MtMFT, Medtr8g106840).

Figure 2. The expression pattern of GmMFT in different tissues/organs.

Stages were defined as follows: Unifoliolate, unifoliolates fully opened; Trifoliolate, from 1^st trifoliolates fully opened to onset of flowering; Flowering, onset of flowering; Podding, initiation of pod growth. Abbreviations for tissues/organs: unifoliolate (U), cotyledon (Cot), shoot apex (SA), trifoliolate (T), pod without seeds (P). Tx means x^th trifoliolate. Tx-U represents unifoliolate at the x^th trifoliolate stage. For example, T3-U means unifoliolate at the 3^rd trifoliolate stage. Similarly, Tx-Tx represents x^th trifoliolate at the x^th trifoliolate stage. Three genes (ACT11, UKN1and UKN2) in combination were used for internal control. Error bars denoted SD.

Figure 3. The expression pattern of GmMFT in developmental and germinating seeds.

A, The GmMFT transcripts in seeds during seed development. Seed development was divided into 12 stages (S1–S12) based on the seed length as previously reported. B, The GmMFT transcripts in seeds during seed germination. Three genes (ACT11, UKN1and UKN2) in combination were used for internal control. Error bars denoted SD.

Figure 4. The expression pattern of GmMFT in germinating seeds with the treatments by exogenous ABA and GA3.

A, The germination rates of soybean seeds under 10 µM ABA or 5 µM GA3 treatment; B, Relative expression of GmMFT in soybean seeds at 12 h, 24 h after treatment with 10 µM ABA or 5 µM GA3. Three genes (ACT11, UKN1and UKN2) in combination were used for internal control. A significant difference in comparison with the “Control” 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 SD.

Figure 5. Germination phenotype of 35S::GmMFT transgenic Arabidopsis seeds.

A, Germination phenotype of 35S::GmMFT transgenic lines in Col background on 1/2 MS medium; B, Germination phenotype of 35S::GmMFT transgenic lines in mft-2 background on 1/2 MS medium; C, Germination phenotype of 35S::GmMFT transgenic lines in mft-2 background on 1/2 MS medium supplemented with 10 µM ABA. A significant difference in comparison with the “Col” for figure 5A or the “mft-2” for figure 5B and figure 5C 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.

Figure 6. The relative expression of germination-related genes in 35S::GmMFT transgenic Arabidopsis seeds.

The seeds at 12-qPCR. Three genes (CSY3, At2g20000, At2g04660) in combination were used for internal control. A significant difference in comparison with the “Col” 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 SD.

Figure 7. Subcellular localization of GmMFT and N-truncated GmMFT.

YFP represents yellow fluorescent signals of GmMFT:YFP, N-truncated GmMFT:YFP or AtMFT:YFP; CFP represents cyan fluorescent signals of the nuclear protein marker AHL22; Chlorophyll represents chloroplast auto-fluorescence; Bright field represents images from white light; Merge represents merge images of the former four images.