To simultaneously suppress the expression of all three subunits of β-conglycinin we constructed an RNAi cassette by the following procedure. First, the intron from pKannibal (Wesley et al., 2001) was amplified employing the primer pair: 5′ primer XbNdXhINTRi (5′-TCTAGA ACATATGGTCCTCGAGAGTTACTAGTACCCCA-3′) containing XbaI, NdeI and XhoI sites (bold) and 3′ primer INTRiRVSXb (5-TCTAGAGGTCGACCGGATATCCGCTTTGTTATATTAGC-3) containing XbaI, SalI, and EcoRV sites (bold). The amplified intron product was cloned into pGEM-T easy to create pKINTRi. The intron was excised by digestion with XbaI and cloned into pHK vector. Primer pair SNd2AplhaRi (5′-GTCGACCATATGTACAGGAACCAAGCATGCCAC-3′; introduced restriction sites SalI and NdeI are underlined and bold, respectively) and Alpha′314XhRV (5′-GATATCCTCGAGTGAGGTTGGTGTGGGCGTGGG-3′; introduced restriction sites EcoRV and XhoI are underlined and bold, respectively) was used to amplify a 314 bp coding region of α′-subunit of the β-conglycinin. This 314 bp product was cloned into pGEM-Teasy vector (Promega, WI) and named as pBCON and sequenced at the University of Missouri DNA Core Facility. DNA prepared from pBCON was subjected to restriction enzyme digests with NdeI and XhoI or EcoRV and SalI to facilitate cloning the 314 bp fragments in the opposite orientation. The NdeI and XhoI excised fragment was ligated into the 5′ end of the pHK-intron while the EcoRV and SalI fragement was ligated into the 3′ end pHK-intron. This procedure resulted in a cassette containing a 314 bp α′-subunit of the β-conglycinin hairpin flanking the pHK-intron. Following digestion with XbaI the entire β-conglycinin hairpin was cloned into the corresponding restriction site of pZPlapha′P binary vector (Kim and Krishnan, 2004). This final vector places the β-conglycinin hairpin under the regulatory control of the α′-subunit of β-conglycinin promoter and the terminator of the potato proteinase inhibitor gene (PinII). This vector also contains the bar-coding region under the regulatory control of cauliflower mosaic virus 35S promoter and 3′-region of the nopaline synthase gene (Figure 1). The final RNAi construct was mobilized into Agrobacterium tumefaciens (strain EHA105) by triparental mating (Friedman et al., 1982). Soybean cultivar Maverick was transformed by Agrobacterium-cotyledonary node method (Hinchee et al., 1988). Regenerated transgenic soybean plants were screened for tolerance to herbicide Liberty by a leaf-painting assay as described earlier (Zhang et al., 1999). The knockout of the target proteins in glufosinate-resistance plants was confirmed by PCR and SDS-PAGE. For all comparisons soybean cultivar Maverick was used and referred as wild-type.

Total RNA was extracted from transgenic soybean seed at the mid-maturation stage (seed size 8–10 mm) using a Trizol reagent (Invitrogen, Grand Island, NY, USA) following the manufacture's protocol. RNA was quantified by measuring the A260/A280 ratio using a spectrophotometer. One μg of DNase I treated RNA was used as template for RT-PCR. Primers used for alpha′ subunit were Alpha′F, 5-ATGATGAGAGCGCGGTTCCCATTACTG-3 and Alpha′R, 5-TCAGTAAAAAGCCCTCAAAATTGAAGAC-3. Primers used for alpha subunit were Alpha F, 5-ATGATGAGAGCACGGTTCCCATTACTG-3 and Alpha′R. Primers used for beta subunit were BetaF, 5-ATGATGAGAGTGCGGTTTCCTTTGTTGG-3 and BetaR, 5- TCAGTAGAGAGCACCTAAGATTGAAG-3. Primers used for glycinin 4 were SoyGy4F, 5- ATGGGGAAGCCCTTCACTCTCTCTCTTTC-3 and SoyGy4R, 5- TTATGCGACTTTAACACGGGGTGAGC-3. The cycling condition of RT-PCR were 50°C for 30 min for cDNA production, 95°C for 15 min for inactivation of cDNA production, then 35 cycles of 94°C for 60 s, 60°C for 60 s, 72°C, for 90 s with a final 72°C for 240 s extension steps.

Soybean protein content was measured using the Leco model FP-428 nitrogen analyzer (LECO Corporation, Michigan, USA). The oil content was quantified by near-infrared reflectance (NIR) spectroscopy (Tecator AB, Hoganas, Sweden). The fatty acid profiles of soybean were determined by gas chromatograph as described previously (Lee et al., 2009). Briefly, crushed seeds were extracted overnight with 5 mL of chloroform: hexane: methanol (8:5:2, v/v/v). Fatty acids from 100 μL aliquots of the extract were methylated with 75 μL of methanolic sodium methoxide:petroleum ether:ethyl ether (1:5:2, v/v/v). Fatty acids were separated utilizing Agilent Series 6890 capillary gas chromatograph (Palo Alto, CA, USA) that was fitted with an AT-Silar capillary column (Alltech Associates, Deerfield, IL, USA). Standard fatty acid mixtures were used for determining relative amounts of each fatty acid. Four replicates of “Maverick” (MAV) and β-conglycinin knockdown line (SAM) were compared using the t test function within JMP® software Version 9 (SAS Institute Inc., Cary, NC). Significantly different means are indicated by ^*, ^**, or ^*** (p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively) and insignificant differences are indicated by “NS.”

Total soybean seed proteins were extracted from 10 mg of dry seed powder with 1 ml of SDS sample extract buffer (2% SDS, 60 mM Tris-HCl, pH 6.8, 5% β-mecaptoethanol), followed by boiling at 100°C for 5 min. After maximum speed centrifuge the supernatant was used for total seed protein fraction. The total seed protein fraction was electrophoresed with 8 or 15% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. For western blot analysis, the total seed proteins were transferred to a nitrocellulose membrane after resolved by SDS-PAGE. After blocking with TBS (10 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing 5% non-fat dry milk, the membrane was incubated over-night with 7S globulin storage protein or the 11 kDa δ-zein antibodies (Kim and Krishnan, 2003) that had been diluted 1:5000 in TBST (TBS with 3% non-fat dry milk containing 0.2% Tween 20). After several washes with TBST, the membrane was incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate. Immunoreactive polypeptide signals were detected by according to the SuperSignal West Pico kit's instruction (Pierce, Rockford, Il, USA).

Two-dimensional gel electrophoresis of soybean seed proteins were performed as described earlier (Krishnan et al., 2009). Coomassie stained gels were destained with multiple changes of ultrapure water to remove background and scanned using an Epson Perfection V700 scanner controlled through Adobe Photoshop. Images were analyzed for proteome differences using Delta2D image analysis software. Delta2D parameters were set to maximize spot detection using global image warping and exact spot matching. Background subtraction parameters were identical for all gels and eliminated a large percentage of spots that were less than 0.05% of the total normalized spot volumes. A total of 732 remained on the fusion image of all gels included in the analysis, after background subtraction, and were used for the total volume ratio normalization. A total of 34 spots were chosen as a subset of this total, for major seed storage protein quantification, comprising nearly 50% of the total volume ratio normalized spot volume. This subset of 2-D resolved proteins were previously identified (Krishnan et al., 2009; Krishnan and Nelson, 2011) using peptide mass fingerprinting (MALDI-TOF MS) and then categorized as shown in Figure 5. The exact same spot regions were chosen from software generated fused image of all gels used in the analysis. Spot % volume quantities were calculated within each comparison individually; Maverick (n = 3) and SAM (n = 3).

Soybeans were grown in an environmentally controlled growth chamber at 26 ± 2°C and 50% humidity. The plants received 14 h of daylight for the duration of the experiment. Each individual plant was grown in its own modified hydroponics container, consisting of a pot containing a 1:1 mixture of perlite:vermiculite, with a lower 2 L reservoir for the nutrient solution. Cotton wicks were placed in the lower portion of each pot to deliver nutrient solution to the plant prior to the root growth eventually reaching the lower reservoir. The nutrient solution composition was: 1.25 mM Ca(NO₃)₂ 4H₂O, 1.25 mM KNO₃, 0.25 mM KH₂PO₄, 0.5 mM Fe-EDTA, 0.5 mM NH₄NO₃, and a 1/1000th addition of a micronutrients mixture (H₃BO₃, 2.86 g L⁻¹; MnCl₂ 4H₂O, 1.81 g L⁻¹; ZnCl₂, 0.095 g L⁻¹; Cu(NO₃)₂ xH₂O, 0.047 g L⁻¹; H₂MoO 4H₂O, 0.09 g L⁻¹). The sulfate control solution contained 0.5 mM MgSO₄ 7H₂O, and the sulfur-rich solution contained 2.0 mM MgSO₄ 7H₂O. The nutrient solution was replaced every 3 days to maintain a consistently fresh nutrient supply. Seeds were harvested approximately 90 days after treatments began.

Amino acid analysis was performed at the Donald Danforth Plant Science Center Proteomics and Mass Spectrometry Facility. Free amino acid content was determined following the protocol of Hacham et al. (2002). For the determination of the total amino acid content the seed powder was first subjected to hydrolysis with 6N HCl. For the quantification of methionine and cysteine, duplicate samples were first subjected to an initial oxidation step using performic acid prior to acid hydrolysis. Amino acids were quantified using the manufacturer's instructions of the Waters AccQ-Tag Ultra Kit on an Acquity UPLC system. Samples were run in quadruplicate and subjected to appropriate statistical analysis.

Greenhouse-grown soybean seeds at R6 stage (Fehr et al., 1971) were cut into several pieces and fixed in FAA (10% formaldehyde, 50% ethyl alcohol, and 5% glacial acetic acid) for 8 h at room temperature. The tissue was dehydrated in a graded ethanol/xylene series and infiltrated with paraffin. Sections were cut with a microtome and processed for immunostaining following the protocol described earlier (Bilyeu et al., 2008). Sections were separately incubated with 1:1000 diluted antibodies raised against the β-subunit of β-conglycinin or Kunitz trypsin inhibitor. Following this step, the sections were treated sequentially with biotinylated linker, streptavidin conjugated to horseradish peroxidase, and substrate-chromogen solution (DAKO). The sections were examined under bright field optics.

Mature dry seeds were surface sterilized by first sequentially incubating with 95% ethanol for 5 min and with 50% commercial bleach for 5 min. Following extensive washes in distilled water the seeds were transferred to 1% water agar plates and germinated in a 30°C incubator for 12 h. Seeds were sliced into several 2–4 mm cubes and fixed for 4 h in 2.5% glutaraldehyde buffered at pH 7.2 with 50 mM sodium phosphate. After several rinses in sodium phosphate buffer the seeds were post-fixed for 1 h with 1% aqueous osmium tetroxide. The seed tissue was dehydrated in a graded acetone series and infiltrated with Spurr's resin. Thin sections of the seed tissue were cut with a diamond knife and collected on 200 mesh copper grids. The grids were stained with 0.5% uranyl acetate and 0.4% lead citrate and examined at 80 kV under JEOL 1200 EX (Tokyo, Japan) transmission electron microscope.

The suppression of the 7S globulins accumulation in soybean seeds was accomplished by RNAi utilizing Agrobacterium-mediated cotyledonary node transformation protocol (Hinchee et al., 1988). Seeds from several independent transgenic lines were screened for their protein composition by SDS-PAGE and immunoblot analysis using antibodies raised against the β-subunit of β-conglycinin (Figure 2). An examination of the Coomassie stained gel revealed that in 50% of the transgenic plants no significant reduction in the accumulation of β-conglycinin had occurred when compared to the wild-type plant (Figure 2A). However, four transgenic RNAi lines showed a complete absence of the β-conglycinin accumulation in their seeds (Figure 2A). The lack of β-conglycinin in these RNAi lines was confirmed by immunoblot analysis using β-conglycinin antibodies (Figure 2B). These four lines were grown for five generations. Twenty individual T5 seeds from each transgenic line were subjected to Western blot analysis. Transgenic plants where all tested seeds failed to accumulate the β-conglycinin were considered to be homozygous plants. These homozygous transgenic RNAi plants are referred as SAM from this point onwards.

We investigated the mRNA levels of the major seed storage proteins between the RNAi line SAM and the wild-type control by RT-PCR. When primers specific for gy1, gy2, gy3, and gy4 were used in RT-PCR reactions, gene-specific products were amplified in both plants (Figure 3A). However, when primers specific for each of the three subunits of β-conglycinin were employed, RT-PCR products were seen only in the wild-type control and not in SAM (Figure 3B). The absence of the β-conglycinin accumulation in SAM seeds was further examined by immunocytochemical localization. Sections of soybean seed were incubated with either β-conglycinin or Kunitz trypsin inhibitor specific antibodies followed by incubation with streptavidin conjugated to horseradish peroxidase. Antibodies raised against the β-conglycinin did not detect their accumulation in the paraffin-embedded sections as evidenced by the absence of brown localization signal (Supplemental Figure 1A) while it was readily detected in the wild-type control seeds (Supplemental Figure 1B). In contrast, when the paraffin sections were incubated with Kunitz trypsin inhibitor specific antibodies positive reactions were seen in both SAM and the wild-type control seeds (Supplemental Figures 1C–D).

To investigate if knockdown of β-conglycinin resulted in any alterations of the seed components we first measured the total protein and oil content of these seeds. Both the wild-type control and SAM contain very similar concentrations of total protein and oil (Table 1). Total amino acid content was significantly higher in β-conglycinin knockdown line (Table 1). Analysis of the five major fatty acids in these seeds by gas chromatograph also showed significant differences. The concentration of palmitic, linoleic and linolenic acids was higher in the wild-type while oleic acid content was higher in SAM (Table 1).

Previous studies have shown that soybean mutants lacking the seed storage proteins accumulate high levels of free amino acids (Takahashi et al., 2003; Schmidt et al., 2011). To examine if similar situation also occurred in the β-conglycinin knockdown soybean line we determined the total and free amino acid content (Figure 4). An examination of the total amino acid composition revealed a slight increase in several of the amino acids in SAM when compared to that of wild-type control (Figure 4A). In contrast, a comparison of the free amino acid composition revealed significant increases in the concentration of several amino acids in SAM seeds especially glutamic acid, aspartic acid, asparagine, arginine, and alanine (Figure 4B). In previous studies arginine represented the main amino acid that contributed to the accumulation of high levels of free amino acids (Takahashi et al., 2003; Schmidt et al., 2011). However, in our studies we observed that arginine was not the most abundant amino acid in the overall free amino acid pool in SAM. Few other amino acids (glutamic acid, aspartic acid, asparagine, and alanine) also contributed significantly to the elevated levels of free amino acid content in SAM seeds (Figure 4B).

Two dimensional-gel analysis was performed to evaluate the changes in the protein composition of RNAi soybean line with that of the wild-type control (Figure 5). This analysis clearly demonstrated that β-conglycinin knockdown RNAi line lacked all the three subunits (α′, α, and β-subunits) of β-conglycinin. Previous studies have shown that suppression of the seed 7S and 11S seed storage proteins resulted an increase in the accumulation of lipoxygenase, sucrose binding protein, basic 7S globulin, Kunitz trypsin inhibitor, soybean lectin, Gly m Bd 30k, Glc-binding protein and seed maturation-associated protein in these seeds (Takahashi et al., 2003; Schmidt et al., 2011). To see if similar changes are also occurred in SAM 2D fractionated soybean seed proteins from SAM and wild-type control (run in triplicate) were analyzed with high-resolution image analysis software (Delta 2D). This analysis indicated that absence of the three subunits of β-conglycinin was associated with an increase in the glycinin content (Figure 5). A comparison of seed protein profiles between SAM and wild-type control did not reveal any substantial differences between other seed proteins (Figure 5).

We had earlier generated transgenic soybean plants expressing 11 kDa δ-zein under the control of the β-conglycinin promoter (Kim and Krishnan, 2004). Since the β-conglycinin promoter was also used for suppressing the expression of β-conglycinin we wanted to use another seed-specific promoter to express the 11 kDa δ-zein. For this purpose a plasmid consisting of soybean glycinin promoter, the coding region of the 11 kDa δ-zein, the 3′ region of the potato proteinase inhibitor gene, together with the cassette containing bar herbicide resistance gene was constructed (Figure 1) and introduced into soybean cv. Williams 82 as described earlier (Kim and Krishnan, 2004). The accumulation of the 11 kDa δ zein in five independent transgenic events was verified by western blot analysis using antibodies raised against the purified 11 kDa δ-zein (Figure 6). The 11 kDa δ-zein expressing soybean lines were grown in the greenhouse for another three generations to produce T4 plants.

Earlier studies have shown proteome rebalancing in seeds can be exploited for elevated expression of foreign proteins (Goossens et al., 1999; Tada et al., 2003; Schmidt and Herman, 2008). We wanted to test if the δ-zein introgression into the β-conglycinin knockdown RNAi line would enhance the accumulation of the δ-zein. For this purpose, crosses were made between homozygous 11 kDa δ-zein expressing soybean plants and β-conglycinin knockdown RNAi soybean plants. Seeds from successful crosses were germinated and a small segment of the seed was assayed by immunoblot analysis for the absence of β-conglycinin and the accumulation of 11 kDa δ-zein. Seeds with the desired combination were grown in the green house and subjected to recurrent selection until homozygous plants with the desired traits were obtained.

The accumulation of the 11 kDa δ-zein in the β-conglycinin knockdown RNAi soybean plant was examined by immunoblot analysis. The 11 kDa δ-zein was readily detected in both original transgenic soybean plants and in β-conglycinin knockdown RNAi background. However, the 11 kDa δ-zein did not accumulate at higher amounts in soybean seeds lacking β-conglycinin, indicating that proteome rebalancing did not enhance the accumulation of the 11 kDa δ-zein.

It has been shown that availability of methionine and cysteine in legumes is the major limiting factor in enhancing the sulfur content of legumes (Tabe et al., 2002). We therefore, examined if the accumulation of the 11 kDa δ-zein can be promoted by providing the developing plants with sulfate-rich solution, which can be assimilated for sulfur metabolism. For this purpose, we grew the soybean plants in hydroponics under two levels of sulfate. In seeds from plants grown in presence 0.5 mM magnesium sulfate, the accumulation of the 11 kDa δ-zein was higher in wild-type background than in β-conglycinin knockdown background (Figures 7A–B). Interestingly, in seeds from soybean plants grown in presence of 2 mM magnesium sulfate there was a drastic increase in the accumulation of the 11 kDa protein. This difference in the accumulation of the δ-zein was much more striking in β-conglycinin knockdown background (Figure 7B). Densitometer scans of the immunoblots revealed that the seeds from plants grown in presence of 2 mM magnesium sulfate had about 3- to 16-fold increases in the accumulation of the 11 kDa δ-zein in the original and introgressed lines, respectively (Figure 7B). In contrast, the overall protein content of soybean seeds was not affected by sulfate treatment. Soybean plants grown in presence of 2 mM magnesium sulfate had 34.9 ± 0.6% while those grown in sulfate-rich solution contained 35.0 ± 0.4% protein. Our preliminary analysis also revealed that the transgenic soybean plants grown in sulfate-rich solution exhibited 1.2 fold-increase in the total sulfur amino acid content when compared with plants grown in presence of 0.5 mM magnesium sulfate.

Previously we showed that expression of 11 kDa δ-zein results in the formation of endoplasmic reticulum derived protein bodies (Kim and Krishnan, 2004). Immunocytochemical localization studies also confirmed that δ-zein is localized within these protein bodies (Kim and Krishnan, 2004). Since sulfur supplementation resulted in a drastic increase in the accumulation of the 11 kDa δ-zein in transgenic soybean plants, we examined if this change was accompanied by an increase in the number of protein bodies. Thin-sections of soybean seeds expressing the 11 kDa δ-zein in β-conglycinin knockdown background grown in 0.5 and 2 mM of magnesium sulfate were examined by transmission electron microscopy (Figure 8). Electron microscopy observation of thin sections of soybean seeds grown in presence of 0.5 mM magnesium sulfate revealed prominent oil bodies and large protein storage vacuoles, the storage compartment for the native glycinin and β-conglycinin (Figure 8A). In addition few spherical electron-dense spherical protein bodies were also observed in the cotyledonary cells (Figure 8A). An examination of seeds grown in 2 mM of magnesium sulfate revealed the presence of numerous protein bodies (Figure 8B). Interestingly soybean seeds from plants grown in presence of 2 mM magnesium sulfate contained numerous small electron-dense protein bodies within vacuoles (Figure 8C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.