Seeds of Bobwhite wheat (T. aestivum cv. Bobwhite), a model wheat cultivar, were germinated in sterile water for 1 week and then transplanted into a climate-controlled chamber (16 h light/8 h dark cycle at 26°C). Different organs including roots, young stems and young leaves were collected from young seedlings, while stamens, mature stems, mature leaves, flag leaves, endosperms, and pistils were collected from mature wheat plants for organ-specific gene expression analysis. The endosperms were harvested from the seeds at 4, 8, 12, 16, 20, 24, 28, and 32 DAP, respectively, and stored at -80°C.

Total RNA extracted from young wheat seeds (20 DAP) was used to synthesize first-strand cDNAs and to amplify the cDNA of TaFUSCA3. To identify the putative TaFUSCA3 gene in wheat, the sequence of the HvFUSCA3 gene (GenBank No.: AM418838) from Hordeum vulgare was used as a query probe to blast the data library of wheat¹ (Moreno-Risueno et al., 2008). A T. aestivum gene sequence with high identity to the ORF of HvFUSCA3 was identified. The sequence analysis showed that it included a complete ORF sequence, which was detected by ORF Finder². This gene was then amplified from cDNA by the use of degenerate primers (Supplementary Table S1). The PCR product was purified and ligated into pMD18-T simple vector (Takara, Dalian, China), which was sequenced to confirm its veracity.

The FUSCA3 sequences from seven plant species were searched for and compared at the NCBI³ and were used for phylogenetic analysis. The alignment of different sequences was performed by using the software DNAMAN, MEGA (version 5.1), and ClustalX2.0, and the neighbor-joining method was used to construct a phylogenetic tree (Saitou and Nei, 1987).

The coding sequences of TaFUSCA3 and TaSPA (excluding the termination codon) were amplified using primers described in Supplementary Table S1 for transient expression in onion epidermal cells. The PCR-amplified products were then subcloned into the pCAMBIA1303-35S-GFP expression vector and pSPYNE-35S/pUC-SPYNE and pSPYCE-35S/pUC-SPYCE vectors for BiFC. The experiment was performed as described by Walter et al. (2004). Information on the vectors used in our study is listed in Supplementary Table S2.

The assay for analysis of transcription activation properties of TaFUSCA3 was conducted in the AH109 yeast strain. The complete coding sequences of TaTAFUS3 (full-length ORF), and N-terminal (TaFUSCA3-N), B₃ domain (TaFUSCA3-B₃), and C-terminal (TaFUSCA-C) fragments were amplified by PCR using specific primers P17-P20, respectively (Supplementary Table S1).

Binding activity between FUSCA3 and the RY-box of both the At2S3 and Ta1Bx7 gene promoter regions was assayed by using the yeast one-hybrid (Y1H) system (Ouwerkerk and Meijer, 2001). RY-boxes of At2S3 and Ta1Bx7, and mutant RY-boxes of Ta1Bx7^∗ containing two repeat copies were subcloned into the vector pHis2.0 subsequently. The particular process of the experiment was described by Wang et al. (2015).

The ORFs of TaFUSCA3, TaFUSCA3^∗, TaSPA, TaPBF, and TaGAMYB were ligated into the pGADT7 and pGBKT7 vectors, respectively, to create the recombined plasmids (Supplementary Table S2). Of the five ORFs, TaFUSCA3^∗ is a mutated version in which the seventh to the 46th amino acids were removed. For yeast mating, various combinations of different plasmids were transformed into yeast strain Y187 by the PEG/LiAc method.

The DNA-protein-interaction enzyme-linked immunosorbent assay (DPI-ELISA) assay is a versatile and effective method that was used to analyze plant TF-specific binding to DNA in vitro. The target protein TaFUSCA3, which was recombined with GST protein, was expressed in prokaryotic expressive Escherichia coli strain BL21. The short complementary primer sequences of biotinylation and non-biotinylation were synthesized by AuGCT DNA-SYNBiotech, Co., Ltd. (Wuhan, China) (Brand et al., 2010).

The intact expression cassette of TaFUSCA3 containing the 35S promoter of cauliflower mosaic virus and the NOS terminator was amplified by reverse-transcription (RT)-PCR and cloned into the eukaryotic expression vector pBI121 to get the recombinant plasmid pBI121-TaFUSCA3. Subsequently, the original promoter of the GFP gene was replaced by the promoter of the At2S3 gene (GenBank No.: At4g27160) that encodes the 2S albumin storage protein in A. thaliana. The primers are listed in Supplementary Table S1. The wild-type A. thaliana line Col and the mutant line fus3-3 were transformed by using the A. tumefaciens floral dipping technique (Clough and Bent, 1998). The transgenic lines were screened on MS medium containing 50 mg/L kanamycin to generate homozygous lines. PCR and qRT-PCR analyses were performed for molecular identification and to assess transgene expression in each generation of transgenic lines.

Expression patterns of TaFUSCA3 in different organs in wheat were analyzed by qRT-PCR (Bio-Rad, Hercules, CA, United States) using the fluorescent DNA intercalating dye SYBR Green I Master Mix. The first-strand cDNAs were synthesized from the total Poly(A)⁺ mRNA using a FastQuant RT Kit (Tiangen, Beijing, China). The gene-specific oligonucleotide primers for the gene expression analysis were designed in the 3′-UTR region of the gene excluding the B₃ conserved domain and other highly conserved domains. To examine the efficiency and specificity, the primers were used for PCR and the products obtained were verified by sequencing as well as a melting curve analysis from 55 to 99°C. It was confirmed that a single band and peak was obtained for each primer pair. Different concentrations of various templates and primers were used to determine the optimum concentrations, and a control set of reactions was performed without template and with primer pair.

For each sample, three biological replicates were performed. The expression of TaFUSCA3 was normalized to that of Taβ-actin as an internal control, and the data were analyzed based on the comparative 2^-ΔΔCT formula (Livak and Schmittgen, 2001).

β-Glucuronidase histochemical assay was performed with the plant tissues following the protocol of Gui et al. (2011). The samples were incubated at 37°C overnight in GUS staining buffer [100 mM sodium phosphate, 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 0.1% Triton X-100, 10 mM Na₂EDTA and 1 mg/mL X-Gluc] for blue color development. After staining, the sections were rinsed with 70% ethanol for at least 1 h. Photographs were taken under fluorescence microscopy (OLYMPUS DP72, Japan).

Protein samples were electrophoresed on 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore) for detection of the target proteins. The membranes were subsequently blocked by incubation in Tris-buffered saline plus Tween 20 (TBST: 25 mM Tris-HCl, 80 mM NaCl, 0.1% Tween 20) containing 6% fat-free milk overnight at 4°C. The membranes were incubated with the primary antibody (diluted 1:4000, 2 h), washed (10 min × 4 times), and treated with the appropriate secondary antibody for 1 h. After washing the membranes again four times with TBST, immunoreactive bands were visualized by using a chemiluminescence detection system (UVP ChemiDoc-It, Upland, CA, United States). The housekeeping β-actin protein was detected as a control (Li et al., 2012).

All experiments in our study were carried out in triplicate. The statistical software program ORIGIN for Windows version 8.1 was used for statistical analysis of the experimental data. Analysis of variance was used to contrast the statistically significant difference by employing the Student’s t-test procedure. The 5% significant differences (P < 0.05) and 1% remarkably significant differences (P < 0.01) were evaluated. Data are the means ± SD in three independent replicates (n = 3).

The cDNA of TaFUSCA3 containing 1,071 bp was obtained from common wheat by use of an in silico cloning method. The FUSCA gene was designated TaFUSCA3 as it had high sequence homology to FUSCA3 (GenBank No.: AM 418838) from H. vulgare. The ORF of TaFUSCA3 encoded a polypeptide of 288 amino acid residues with a predicted relative molecular mass of 32.3 kDa containing a B3 DNA binding domain. The TaFUSCA3 gene was localized on chromosomes 3B, 3DL, and 3AL, which was also validated by the latest URGI Blast database⁴.

Multiple sequence alignment analysis indicated that TaFUSCA3 had a high sequence identity with the FUSCA3 proteins from some cereal crops (89.3% with HvFUSCA3 from H. vulgare, 76.3% with BdFUSCA3 from Brachypodium distachyon, and 68.47% with OsFUSCA3 from Oryza sativa), while it had lower sequence identity with FUSCA3 proteins from other monocots, such as Triticum urartu, Aegilops tauschii, and Zea mays (64.8, 64.2, and 61.8%, respectively) (Figure 1A). The protein sequences used to perform the multiple sequence alignment are shown in Supplementary Table S3. According to the Multiple EM for Motif Elicitation tool⁵, TaFUSCA3 had seven motifs, including a typical conserved B3 domain (amino acids 64–174), which could recognize and bind to the specific sequence (RY-boxes), and a transcription activating domain (TAD) containing 115 amino acid resides in the C-terminal region.

In order to further confirm the evolutionary relationships between TaFUSCA3 and FUSCA3 proteins from seven other plant species, a phylogenic tree was generated to analyze the historical evolution based on amino acid sequence alignment (Figure 1B). This analysis provided a further indication that FUSCA3 proteins are very highly conserved evolutionarily, and TaFUSCA3 presented the closest genetic relationship with HvFUSCA3. These results showed that TaFUSCA3 from common wheat was a member of the FUS3 family.

In order to analyze the subcellular localizations of TaFUSCA3 and TaSPA, the full-length ORFs of these proteins were fused to GFP and were transiently expressed in onion epidermal cells. As shown in Figure 2, both TaFUSCA3 and TaSPA were localized in the nucleus under the dark field for green fluorescence and the bright field, whereas the GFP alone was distributed throughout the cytoplasm and nucleus.

To define possible functions of TaFUSCA3, organ-specific expression analysis was performed by using qRT-PCR with RNA extracted from different organs including roots, young stems, young leaves, endosperms, mature stems, mature leaves, flag leaves, stamens, and pistils. The expression levels of the TaFUSCA3 gene in different organs revealed a distinctive expression pattern, and the results showed that TaFUSCA3 was expressed in all of the organs and had higher expression in the stamen, followed by the endosperm and pistil (Figure 3). This indicated that TaFUSCA3 could participate in the growth and development of wheat and in endosperm development. In addition, significantly low expression was detected in roots, stems, mature stems, and flag leaves.

In order to further investigate the expression pattern of TaFUSCA3 in the wheat endosperm, real-time qRT-PCR was performed with RNAs obtained from endosperms, which were isolated from the grains at different DAP (4, 8, 12, 16, 20, 24, 28, and 32). As presented in Figure 4A, the transcript level of TaFUSCA3 was increased gradually from 4 DAP and peaked (sixfold) at 20 DAP, then decreased.

The expression of TaFUSCA3 was similar to that of TaSPA (GenBank No.: D78609, a storage protein activator), which encodes a wheat bZIP TF of the O2-like type (Albani et al., 1997). The Pearson correlation coefficient between the expression level of TaFUSCA3 and TaSPA was 0.8670, which indicated that the expression of TaFUSCA3 was correlated positively and significantly with that of TaSPA. The expression pattern of TaFUSCA3 was consistent with that of TaSPA, indicating that TaFUSCA3 might be a regulator of the glutenin in the developing endosperm.

The expression level of the HMW-GS gene (Glu-1Bx) Ta1Bx7 was also examined by real-time qRT-PCR (Figure 4B). Its expression level reached a peak at 16 DAP and then reduced gradually. Wheat grain development has three growth stages: grain enlargement (10–14 days after flowering), grain fill (15–35 days after flowering), and physiological maturity. The seed increases rapidly in size as the cells that enclose the embryo sac divide and amplify during the grain enlargement stage. The grain weight increases at a constant rate as carbohydrate and protein are deposited into the grain during the grain fill stage. The SSP genes are transcribed at peak levels in the early grain fill stage in order to prepare enough mRNA templates for the next stages of carbohydrate and protein accumulation. Transcription of SSP genes is then reduced as grain development gradually comes to an end into the physiological maturity stage. The observation that TaFUSCA3, TaSPA, and Ta1Bx7 presented almost identical expression patterns at each developmental stage suggested that TaFUSCA3 and TaSPA could regulate the gene expression of the HMW-GS Ta1Bx7 in wheat (Shewry and Halford, 2002; White and Edwards, 2008).

TaFUSCA3 could activate the expression of the albumin At2S3 gene in A. thaliana and the glutenin Ta1Bx7 gene in wheat by binding the cis-acting RY-box element (CATGCA) in the promoter specifically (Figure 6). To determine whether it could activate the storage protein promoter, we compared four promoter sequences of SSPs, including At2S3, 1Dx5, 1Bx7, and 1Bx13 and the sequences of them were listed in the Supplementary Table S5. The promoters of albumin At2S3 (GenBank No.: At4g27160) in A. thaliana and the HMW-GS Ta1Bx7 that have eight RY-boxes were isolated and putative control elements were screened by using PLACE⁶ (Geng et al., 2014). Yeast strains transformed with TaFUSCA3 and the RY-box from the promoters of At2S3 and Ta1Bx7 grew well on selective media (Figure 6A), which indicated that TaFUSCA3 could interact with the promoters of At2S3 and Ta1Bx7.

In addition, a DPI-ELISA was performed to confirm the interaction between TaFUSCA3 and the RY-box in the promoter of Ta1Bx7. Firstly, we successfully obtained the TaFUSCA3-GST fusion protein by use of the prokaryotic expressive assay (Figure 5). As presented in Figure 6B, TaFUSCA3 with the RY-box produced a stronger absorption signal than that with the RYm-box, which had remarkably significant difference in absorption (P < 0.01). The competition experiment by DPI-ELISA was performed to verify the interaction by appending different concentrations of non-biotinylated RY-probe to the binding reaction (Figure 6B). The increasing concentrations of non-biotinylated RY-probes competed with the binding of TaFUSCA3 to the biotinylated RY-probe successfully. The absorption signal was significantly decreased with the addition of 10 pmol non-biotinylated RY-probe (P < 0.05) and it was remarkably significantly reduced when the concentration of non-biotinylated RY-probe was increased up to 50 pmol (P < 0.01). However, the mutated RYm-probe rarely competed with the recognition of TaFUSCA3 to the biotinylated RY-probe, even at increased concentrations. Hence, specific recognition between TaFUSCA3 and the RY-box of the promoter of Ta1Bx7 was confirmed by the Y1H and DPI-ELISA assays.

Transcriptional activity of the TaFUSCA3 protein was analyzed by using the Y1H system. In order to verify which domain determines the activation capacity, yeast strain Y187 was transformed with the recombinant plasmids containing full-length TaFUSCA3, the N-terminal region (1st to 60th amino acids), the C-terminal region (172th to 288th amino acids), and the B3 domain (61th to 171th amino acids) combined with the GAL4 binding domain, respectively. The growth status of the strain was observed on selective medium (Figure 7A). The yeast cells transformed with the BD-TaFUSCA3 and BD-C-terminal constructs grew well on SD medium without tryptophan, adenine, or histidine, demonstrating that TaFUSCA3 acted as a transcriptional activator. In β-galactosidase activity assay, the β-galactosidase activity of the C-terminal region was 1.4 times that of the whole protein; the other domains did not grow on the selective medium (Figure 7B). These results indicated that the full TaFUSCA3 and its C-terminal region had transcriptional activities.

To define the interaction of the TFs related to the regulation of wheat storage protein expression, additional cDNAs of TaSPA and TaPBF and TaGAMYB were cloned (Supplementary Table S4) and the Y2H assay was performed (Hu et al., 2013). For this purpose, we constructed plasmids AD-FUSCA3, AD-FUSCA3^∗, and AD-SPA. In the meantime, we constructed effector plasmids BD-FUSCA3, BD-SPA, BD-PBF, and BD-GAMYB, in which the TFs were fused to the binding domain of the Gal4 TF. Co-transfections into the Y187 yeast strain were performed, and the resulting transformants were tested on SD/-Lea-Trp. As shown in Figure 8A, yeast cells harboring AD-FUSCA3, BD, or plasmids without inserts (pGBKT7 and pGADT7) did not show β-galactosidase enzyme activity. While the β-galactosidase enzyme activity of the combination of AD-FUSCA3 and BD-SPA was remarkably significantly increased compared with that of AD-FUSCA3 with BD-PBF and AD-FUSCA3 with BD-GAMYB (P < 0.01). Growth was not found in the mutant FUSCA3^∗ (Figure 8B). Co-expression of AD-SPA with BD-FUSCA3 resulted in very high levels of β-galactosidase, and the enzyme activity of the combination of AD-FUSCA3 and BD-SPA was roughly twofold higher than that of the group for AD-FUSCA3^∗ and BD-SPA (P < 0.01), which implied that there exists an interaction between FUSCA3 and SPA.

The β-galactosidase activity assay further confirmed interactions between SPA and the N-terminal domain of FUSCA3 in yeast (Figure 8C). TaSPA was used as prey to interact with the N-terminal domain, B3 domain, and C-terminal domain from TaFUSCA3. For the N-terminal domain, the interaction with SPA was roughly fourfold greater than that of the B3 domain (P < 0.01). Because the C-terminal domain had a transcriptional activation activity, it showed a higher β-galactosidase activity when used as bait. But it presented a lower enzyme activity when used as prey.

To further confirm the interaction between TaFUSCA3 and TaSPA, a BiFC assay was performed in epidermal cells of onions. The C-terminal fragment of yellow fluorescent protein (YFP) fused with TaFUSCA3 was able to interact with the N-terminal fragment of YFP fused with TaSPA in the nucleus (Figure 8D). The fluorescence signal was found only in the nucleus. These results indicated that TaFUSCA3 interacts with TaSPA and the complex is colocalized in the nucleus.

To verify the inference about conservation of function between TaFUSCA3 and AtFUSCA3, the A. thaliana wild-type Col and mutant type fus3-3 were transformed with TaFUSCA3 (Keith et al., 1994). The expression cassette including CDS (coding sequence) of TaFUSCA3, 35S promoter, and NOS terminator was inserted into pBI121 in which the GFP gene was driven by the promoter of At2S3. The results of PCR and qRT-PCR for the molecular identification and detection of transgene expression in the transgenic lines are shown in Supplementary Figures S1, S2. The expression level of albumin 2S had been confirmed to be strongly decreased in the grains of the FUSCA3 mutant line (Luerssen et al., 1998). The mature seeds of wild-type Col and mutant type fus3-3 transformed respectively with the GFP gene under the control of the At2S3 promoter and TaFUSCA3 were observed by fluorescence inversion microscopy (OLYMPUS DP72, Japan). As shown in Figure 9A, fluorescence intensity of GFP, which was weakened intensively in the fus3-3 lines, was recovered to nearly the same level as that of the wild-type in the grains expressing TaFUSCA3 constitutively.

The transient expression of TaFUSCA3 in onion epidermal cells was used to confirm the transcription activation of the Ta1Bx7 gene promoter (Figure 9B). The GUS staining of the onion epidermal cells revealed that expression level of GUS driven by the Ta1Bx7 promoter was obviously increased following co-transformation with the TaFUSCA3 gene. It could be inferred that TaFUSCA3 activated the transcription of the Ta1Bx7 promoter, which led to the increase of expression of GUS.