Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman–Birk protease inhibitor and suppresses the accumulation of the β-subunit of β-conglycinin in soybean seeds

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5′-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman–Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC–MS and GC–MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37–52% and 15–19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.


Results
Generation of transgenic soybean plants overexpressing ATP sulfurylase. A plant transformation construct harboring the coding region of soybean ATP sulfurylase 1 without the transit peptide under control of the CaMV 35S promoter was inserted into genome of soybean cultivar Maverick by Agrobacteriumcotyledonary node transformation. This procedure resulted in the regeneration of four independent transgenic events (ATPS1, ATPS2, ATPS3, and ATPS4) that showed resistance to Liberty herbicide. To verify the expression of ATP sulfurylase in these transgenic plants, immunoblot analysis was performed with antibodies raised against soybean ATP sulfurylase. Total protein obtained from young seeds (20 days after flowering) of four independent transgenic soybean lines and a non-transgenic soybean line were resolved by SDS-PAGE (Fig. 1a). Immunoblot results showed strong reaction against a 42 kDa protein from all the four independent transgenic soybean lines while only a faint reaction was detected in the non-transgenic control soybean line (Fig. 1b). To verify the distribution of the introduced ATP sulfurylase, which was expressed in the absence of a transit peptide, we isolated different crude subcellar fractionations by differential centrifugation. Immunoblot analysis revealed ATP sulfurylase was predominantly distributed in the cytosol, although lower amounts were also found in other fractions (Fig. 1c). We also measured the ATP sulfurylase activity in these transgenic soybean lines. When compared to the non-transgenic control soybean line, ATP sulfurylase activity was 2 to 2.5-fold higher in the transgenic soybean lines (Fig. 1d).

Overexpression of ATP sulfurylase alters seed protein content and composition. To investigate
if overexpression of ATP sulfurylase brought about changes in seed components, we measured the total protein and oil content of field grown mature dry seeds. The total protein content of seeds overexpressing ATP sulfurylase (37.1 ± 0.5%) was about 3% lower than that observed in non-transgenic control seeds (40.1 ± 0.6%). In contrast, the oil content of the ATP sulfurylase overexpressing seeds (22.3 ± 0.3%) was about 1% higher than the wild-Scientific Reports | (2020) 10:14989 | https://doi.org/10.1038/s41598-020-72134-z www.nature.com/scientificreports/ type seeds (21.1 ± 0.4%). We also observed differences in seed yield, though statistically non-significant, between  Similarly, a decrease in seed weight was observed in ATP sulfurylase overexpressing plants (13.96 g ± 0.74 g/100 seeds) when compared to the non-transgenic control seeds (16.62 g ± 1.03 g/100 seeds). The decrease of protein content in ATP sulfurylase overexpressing mature seeds prompted us to investigate if this loss was accompanied by changes in the protein composition. For this purpose, we conducted high-resolution 2-D gel electrophoresis of mature soybean seed proteins (Fig. 2). Examination of Coomassie stained 2-D gels revealed several discrete protein spots. We and others have previously identified these 2-D resolved proteins by mass spectrometry [26][27][28] . The two most abundant soybean seed storage proteins are the 7S β-conglycinin and 11S glycinin, respectively. In the wild-type seeds, the three sub-units of β-conglycinin migrated as abundant proteins spots with apparent molecular weight of 76 kDa, 72 kDa, and 52 kDa, respectively (Fig. 2a). The β-subunit of β-conglycinin migrated as five distinct protein spots with slightly different isoelectric points (Fig. 2a). Interestingly, the abundance of these protein spots in ATP sulfurylase overexpressing soybean seeds was drastically reduced compared to the wild-type seeds (Fig. 2b). The other abundant seed storage protein, the glycinin which is made of acidic and basic subunits, resolved into discrete protein spots migrating with apparent molecular weights of 32-34 kDa and 20-22 kDa, respectively (Fig. 2). The abundance of these protein spots was determined by Delta2D image analysis (Supplemental Fig. 1 and Supplemental Table 1). The percent spot volume intensity of some the abundant seed protein spots were relatively higher in ATP sulfurylase overexpressing soybean compared to wild-type plants (Supplemental Table 1). Additionally, several low molecular weight protein spots migrating at 12-14 kDa also accumulated at higher amounts in the ATP sulfurylase overexpressing soybean seeds (Supplemental Table 1).

Immunoblot analysis confirms overexpression of ATP sulfurylase suppresses the accumulation of the β-subunit of β-conglycinin and promotes the accumulation of Bowman-Birk protease inhibitor (BBI).
Our high-resolution 2-D gel analysis indicated that overexpressing ATP sulfurylase in transgenic soybean leads to major alterations in the seed protein profiles ( Fig. 2 and Supplemental Table 1). To confirm these changes and verify the identity of these proteins we performed immunoblot analysis using polyclonal antibodies raised against the β-subunit of β-conglycinin (Fig. 3) and BBI (Fig. 4). SDS-PAGE analysis of the total seed proteins from the dry seeds of four independent transgenic plants clearly showed the absence of a 52 kDa protein (Fig. 3a). In contrast, this protein was prominently visible in the wild-type seed protein extracts. Antibodies raised against the β-subunit of β-conglycinin reacted strongly against this polypeptide, while only a faint reaction can be detected in the transgenic soybean seeds (Fig. 3b).
Since our 2-D gel comparison indicated that the BBI accumulation may be enhanced in the ATP sulfurylase overexpressing soybean seeds, we conducted immunoblot analysis to confirm this observation. Previously, we demonstrated that 50% isopropanol can be used to preferentially extract BBI from soybean seeds 29,30 . Therefore, we extracted 50% isopropanol-soluble proteins from the dry seeds of four independent transgenic plants. SDS-PAGE analysis of 50% isopropanol-soluble proteins revealed similar protein profile between the control and transgenic plants (Fig. 4). A group of proteins migrating with apparent molecular weights of 10-14 kDa were much more abundant in the transgenic seeds (Fig. 4a). Antibodies raised against soybean BBI reacted strongly against the 10-14 kDa polypeptides confirming that these proteins are isoforms of BBI (Fig. 4b). In contrast, www.nature.com/scientificreports/ a longer exposure was required to detect BBI accumulation in wild-type seeds clearly establishing that BBI accumulates several-fold higher in the transgenic soybean seeds compared to non-transgenic seeds (Fig. 4c).

Impact of overexpression of ATP sulfurylase on seed metabolites.
To obtain insight into the biochemical changes resulting from overexpression of ATP sulfurylase, we carried out metabolite profiling of soybean seeds. By employing a combination of metabolite profiling platforms a total of 124 metabolite compounds were detected, quantified and analyzed (Supplemental Table 2). A heat map of metabolite levels in dry  www.nature.com/scientificreports/ seeds of wild-type and ATP sulfurylase overexpressing soybean plants showed significant differences in several metabolites belonging to major biochemical pathways (Supplemental Fig. 3). This includes 38 amino acids and amino acid derivatives, 9 carbohydrates, 63 lipids, 6 cofactors/prosthetic groups/electron carriers, 3 nucleotides, 4 secondary metabolites, and 1 xenobiotic chemical. Out of 124 metabolites analyzed, 84 were increased and 40 decreased in ATP sulfurylase overexpressing seeds compared to non-transformed wild-type seeds (Supplemental Table 2). Figure 5 shows box plot layouts of several metabolites that are significantly elevated in ATP sulfurylase overexpressing soybean seeds. Cysteine was nearly twofold higher in ATP sulfurylase overexpressing seeds than in the wild-type seeds. Similarly, sulfate was also higher in ATP sulfurylase overexpressing seeds. Several N-acetylated amino acids, such as N-acetyltryptophan, N-acetylglutamate, N-acetylglutamine, N-acetyl-3-methylhistidine and N-acetyl-1-methylhistidine were also increased in ATP sulfurylase overproducing seeds (Fig. 5). Overexpression of ATP sulfurylase had a significant effect on lipid metabolism. Several classes of lipids increased in ATP sulfurylase overexpressing seeds relative to the wild type control (Fig. 6). Many of these classes are components of membranes, such as phospholipids, sulfolipids, galactolipids, and sterols, but also included diacylglycerols and monoacylglycerols. The levels of 26 metabolites involved in the amino acid super pathway was significantly lower (p < 0.05) in ATP sulfurylase overexpressing soybean seeds compared to control non-transgenic seeds (Supplemental Table 2). The concentrations of γ-glutamylalanine, γ-glutamylglutamine, γ-glutamylleucine and γ-glutamylvaline, which are related to glutathione metabolism, were noticeably lower in ATP sulfurylase overexpressing soybean seeds. Similarly, modest decreases in TCA cycle metabolites (i.e., citrate, tricarballyate, isocitrate and malate) were also observed in ATP sulfurylase overexpressing soybean seeds (Supplemental Table 2).
Overexpression of ATP sulfurylase improves the sulfur amino acid content of seeds. Because the transgenic soybeans generated in this study exhibited elevated levels of ATP sulfurylase activity, we examine if the overexpression led to an increase in the sulfur amino acid content of the seeds. For this purpose, we quantified protein-bound amino acid content of four independent transgenic plants and compared with the non-transgenic soybean seeds. The amino acid content was determined with the use of high-pressure liquid chromatography (HPLC) ( Table 1). The protein-bound cysteine content (expressed in mole %) in non-transgenic soybean seeds was 1.83% compared to 2.57%, 2.50%, 2.60% and 2.78% in the four independent transgenic soybean plants overexpressing ATP sulfurylase (Table 1). This represents 40.4%, 36.6%, 42.0%, and 51.9% increases in the cysteine content in these ATP sulfurylase overexpressing plants. Similarly, methionine content in non-transgenic soybean seeds was 1.46% compared to 1.71%, 1.69%, 1.68%, and 1.74% in the four transgenic soybean plants overexpressing ATP sulfurylase (Table 1). Arginine, valine and leucine content in all the four ATP sulfurylase overexpressing seeds were lower than in the wild-type seeds (Table 1).

Discussion
Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content of important crops [1][2][3][4][5][6] . In this pathway, ATP sulfurylase catalyzes the formation of APS and inorganic pyrophosphate (PP i ) from sulfate and ATP. The production of APS by ATP sulfurylase is the committed step in plant sulfur assimilation and is responsible for the generation of a high energy phosphosulfate bond that drives subsequent metabolic www.nature.com/scientificreports/ steps in the pathway [15][16][17] . Recent studies have targeted the overexpression of rate-limiting enzymes to accelerate the flux through the entire pathway. For example, serine acetyltransferase and OASS, two key cysteine biosynthesis enzymes, have been overexpressed in different plants including maize, lupin, potato, and rice leading to an increase in the cysteine and/or methionine content 8,9,[31][32][33] . Some of these transgenic plants accumulated toxic intermediates resulting in growth abnormalities 34 . Earlier, we successfully overexpressed cytosolic isoform of OASS in transgenic soybean and observed a 58-74% increase in the protein-bound cysteine compared to wild-type plants 9 . Unfortunately, overexpression of OASS in transgenic soybeans caused growth reduction and impaired nodulation 35 . In the current study, constitutive overexpression of ATP sulfurylase under the control of CaMV promoter resulted in a 37-52% and 15-19% increase in the protein-bound cysteine and methionine content of transgenic seeds. Though the extent of increase the sulfur amino acid content is not as high as in the case of OASS overexpressed transgenic soybean plants, it was still accompanied by a slight growth reduction. Based on these observations, growth retardation observed in transgenic plants due to overexpression of sulfur assimilatory enzymes needs to be overcome to make this approach practicable. Previously, it was demonstrated that overexpression of APS reductase, an enzyme that catalyzes the conversion of APS to sulfite, also resulted in stunted plants; however, this negative effect was completely over come when an unregulated APS reductase was placed under the control of mesophyll-specific PepC or bundle sheath cell-specific RbcS promoter 14 . A similar approach may be required to overcome the negative effect of ATP sulfurylase overexpression in transgenic soybeans.
Metabolite profiling revealed significant changes in N-acetylated amino acids, which were increased in ATP sulfurylase overexpressing seeds relative to wild-type seeds. The reason for this increase is not clear. It seems likely that these compounds arise non-enzymatically, possibly by a reaction with acetyl-CoA. Such reactions are known to occur under relatively basic conditions within cells or in vitro 36 . It is possible that the ATP sulfurylase overexpressing seeds either contained higher levels of acetyl-CoA or that the internal pH or other conditions were more conducive for the reaction to occur. Another interesting aspect that was uncovered by our metabolite analyses relates to lipid metabolism. Several classes of lipids showed significant increase in ATP sulfurylase Figure 5. Metabolites elevated in ATP sulfurylase overexpressing transgenic seeds. Box plots were generated for metabolites that showed significant increase using both t-test and false discovery rate (FDR), with p < 0.05 and q < 0.10 as significant levels. Mav refers to wild-type seeds and ATPS refers to ATP sulfurylase overexpressing transgenic seeds. The numbers on the y-axis refer to relative abundance of metabolites.
Scientific Reports | (2020) 10:14989 | https://doi.org/10.1038/s41598-020-72134-z www.nature.com/scientificreports/ overexpressing seeds levels relative to the wild-type control seeds. Many of these lipids are components of membranes, such as phospholipids, sulfolipids, galactolipids, and sterols. Higher levels of membrane lipids imply that the transgenic seeds harbored more membrane structures than the control seeds. Increased diacylglycerides could result either from degradation of triacylglycerides (which are not measured here) or from new synthesis of phospholipid precursors. The increased amounts of lyso-phospholipids imply some lipolysis and membrane turnover is occurring 37 . The role of all these metabolite changes in transgenic soybean seeds and how it is related to altered flux in sulfur assimilatory pathway need further investigation.
Overexpression of ATP sulfurylase resulted in major changes in the protein profile of the soybean seeds. The two most abundant seed storage proteins of soybean are the 7S β-conglycinin and 11S glycinin 38,39 . Because of their abundance the nutritive value of soybean is mainly dependent on these two group of proteins. The 11S glycinin is relatively rich in sulfur amino acids when compared to the 7S β-conglycinin 40 . The β-conglycinin are glycoproteins and are composed of α′-, α-, and β-subunits. Interestingly the β-subunit of β-conglycinin is totally devoid of both methionine and cysteine 41 and their abundance may lower the nutritive value of soybean seed proteins 42 . In our study, we found that overexpression of ATP sulfurylase resulted in drastic reduction in the accumulation of the β-subunit of β-conglycinin. Interestingly, the accumulation of this subunit is influenced by various factors including hormones, nodulation, sulfur, and nitrogen 42,43 . Nitrogen, sulfur deficiency and exogenous Figure 6. Graphical representation of several classes of lipids in wild type (MAV) and ATP sulfurylase (ATPS) overexpressing transgenic seeds. Box plots were generated for metabolites that showed significant increase using both t-test and false discovery rate (FDR), with p < 0.05 and q < 0.10 as significant levels. The numbers on the y-axis refer to relative abundance of metabolites.
Scientific Reports | (2020) 10:14989 | https://doi.org/10.1038/s41598-020-72134-z www.nature.com/scientificreports/ application of O-acetylserine, a cysteine precursor, to immature cotyledons promoted the accumulation of the β-subunit of β-conglycinin. In contrast, the accumulation of this subunit was drastically lowered when soybean plants were exposed to methionine or glutathione 44,45 . In our study, ATP sulfurylase overexpressing plants contained significantly higher amounts of cysteine and methionine. The increase in the methionine availability in transgenic seeds may be a contributing factor for the suppression of the β-subunit of β-conglycinin accumulation observed in our study. A recent study reported a 31.5% increase in the sulfur amino acid content in soybean lines that tend to accumulate low level of the β-subunit of β-conglycinin 46 . Transcriptome analysis of this soybean line revealed up regulation of genes involved in anabolism of cysteine, methionine and glutathione indicating a close relationship between sulfur amino acid content and the β-subunit of β-conglycinin accumulation 46 . Previously, we reported transgenic soybean plants overexpressing a cytosolic OASS contained higher amounts of BBI 9 . Similarly, in this study we have demonstrated that overexpression of ATP sulfurylase also causes a dramatic increase in the accumulation of BBI. OASS, a member of the β-substituted alanine synthase enzyme family, is the last committed enzyme in the cysteine biosynthetic pathway, while ATP sulfurylase is the first enzyme in the sulfur assimilatory pathway. Overexpression of either of these two enzymes leads to elevated levels of cysteine. Soybean seeds responds to excess cysteine by promoting the accumulation of BBI, a protein that contains 14 cysteine residues 47,48 . The elevated levels of BBI in these transgenic plants, though significantly increases the overall sulfur-amino acid content of the seeds, may limit its utilization in the animal feed. Since protease inhibitors are major anti-nutritional factors preventing protein digestion, emphasis should be placed to not only reduce the inhibitory activity but also to maintain or elevate the sulfur-containing amino acid content of the seeds. An earlier study has shown that it is possible to generate soybean seeds with lower BBI activity by expressing inactive form of the inhibitor gene in transgenic soybeans 49 .
The relative abundance of BBI can be used as an indicator of the sulfur amino acid content of soybean seed proteins and can be exploited as an indirect measurement of cysteine content. Currently, HPLC is used to measure the cysteine and methionine content. However, this method is time-consuming, expensive, and not amenable for high-throughput screening. As an alternative approach, cysteine content of soybean seeds can be indirectly measured by ELISA using antibodies raised against BBI. This approach will enable high-throughput screening of thousands of soybean lines and enable identification of potential soybean cultivars with high sulfur amino acids.
To date, efforts to alter sulfur amino acid content in various plants, including maize, rice, and soybean, have resulted in increased cysteine and/or methionine levels, but also a range of other effects on plant metabolism, seed composition, and plant growth 8,9,13,14,[31][32][33]35,[50][51][52][53] . As shown here and in previous work 46 , increased expression of either sulfur assimilation (ATP sulfurylase) or cysteine biosynthesis (OASS) enzymes in soybean enhance sulfur-containing amino acid content but with rebalanced distribution of sulfur-rich seed proteins. Similar approaches that enhancement of sulfur metabolism in maize led to increases of methionine-rich zein seed proteins without negative effects on plant growth 13,14 , which highlights differences in nutrient metabolism and seed development in different crop plants. Plant growth relies on source-sink relationships, along with associated control mechanisms; however, these vary both by nutrient and by plant. Importantly, the plasticity of amino acid source-sink relationships is critical to allowing for compositional changes in seeds for survival of the next generation 52 . Yet, how targeted changes in plant metabolism, such as overexpressing a key enzyme like ATP sulfurylase in the sulfur assimilation pathway, alters metabolome and/or proteome plasticity remains to be understood in a predictive fashion.

Materials and methods
Plasmid construction. We previously reported the cloning and sequencing of Glyma10g38760a, a cDNA that codes for soybean ATP sulfurylase isoform 1 23 . The coding region of soybean ATP sulfurylase isoform 1 lacking the plastid localization sequence (GmATP SULFURYLASEΔ48) was amplified from the cDNA clone with appropriate primers 23 . To the amplified DNA, BamHI and NotI restriction sites were introduced for facilitation of cloning into an intermediate vector resulting in plasmid pATPS1. Following this step, the insert from the intermediate plasmid pATPS1 was digested with BamHI and XbaI and then cloned into the corresponding sites of pZ35S1, resulting in pZATPS1. The final plant transformation vector consisted of cauliflower mosaic virus 35S promoter (CaMV 35S), the ATP sulfurylase coding region, together with the cassette containing the CaMV 35S promoter, the bar-coding region and the 3′-region of the nopaline synthase gene. The soybean transformation vector pZATPS1 was transferred of into Agrobacterium tumefaciens (strain EHA105) by triparental mating.

Production of transgenic soybean lines overexpressing ATP sulfurylase. Transgenic soybean
plants were generated at the Plant Transformation Core Facility at the University of Missouri, Columbia. Agrobacterium-cotyledonary node transformation was used to transform Soybean cv. Maverick and regenerated transgenic soybean plants were screened for tolerance to herbicide Liberty by a leaf-painting assay. DNA from plants showing resistance to the herbicide was used for PCR to verify the integration of the introduced ATP sulfurylase. The overexpression of ATP sulfurylase from four independent transgenic soybean lines was verified by immuno-blot analysis and by measuring ATP sulfurylase activity, as detailed below. Determination of oil and protein content. Mature seeds harvested from field-grown homozygous transgenic soybean plants and non-transgenic control plants were used for the determination of protein and oil content. Near-infrared reflectance spectroscopy (Tecator AB, Hoganas, Sweden) was used to quantify oil content. Protein content was quantified using the LECO Model FP-428 nitrogen analyzer.
One-dimensional and two-dimensional SDS-PAGE. For extraction of total proteins 10 mg of finely ground dry soybean seed powder was placed into a 2 ml Eppendorf tube containing with 1 ml of 1X SDS sample buffer (0.06 M Tris-HCl, pH 6.8, 2% SDS (w/v), 10% glycerol (v/v), and 5% 2-mercaptoethanol (v/v). In the case of developing seeds, 100 mg of seed tissue was used for total protein extraction. The contents of the tubes were vigorously agitated on a vortex followed by centrifugation at 16,100 × g for 10 min at room temperature. The resulting supernatant was transferred to a new plastic tubes and placed in a boiling water bath for 5 min. A small aliquot (10 µL) were routinely used to separate the total proteins by electrophoresis. Protein separation on 1-D gels (13% SDS-PAGE), was at a constant current of 20 mA/gel in a Mini250 gel apparatus (GE Healthcare). Separated proteins were visualized by staining the gels in Coomassie R-250. 2-D gel electrophoresis was carried out as previously described 54 . After electrophoresis, gels were immediately removed and fixed in 5:4:1 (methanol:water:acetic acid) solution for 1 h, followed by two brief rinses in ultrapure water, then stained in a Coomassie G-250 solution for overnight.
Image acquisition and analysis. 1-D and 2-D Coomassie-stained gels were destained with multiple changes of water to remove background. Gels were scanned using an Epson V700 Perfection scanner controlled through Adobe Photoshop. Delta2D software (Decodon GmbH, Greifswald, Germany) was used for differential image analysis and provided the % protein spot volume data for comparison. Delta2D default parameters were set to maximize spot detection using global image warping and exact spot matching within and between groups (Group 1, Maverick, n = 3; Group 2, ATPS, n = 3). Percent spot volume values were determined for a select group of proteins and error is reported as the standard error of the mean.
Immunoblot analysis. SDS-PAGE separated proteins were electrophoretically transferred to nitrocellulose membranes as previously described 54 . Antibodies raised against soybean ATP sulfurylase 23 , soybean OASS 55 , and soybean Bowman-Birk protease inhibitor (BBI) 9 , were used for immunoblot analysis at 1:10,000 dilution. The β-subunit of β-conglycinin antibody 56 was used at 1:100,000 dilution. Following the incubation with the antibodies, the nitrocellulose membranes were washed in three changes of TBST (0.01 M Tris-HCl, pH 7.5, 0.5 M NaCl and 0.2% Tween 20) and incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad) that had been diluted at 1:20,000. Immunoreactive proteins were detected utilizing a Super Signal West Pico chemiluminescence kit (Pierce).
In order to localize the ATPS activity in soybean plants, freshly harvested leaf material was subjected cell fractionation following established protocol 57 . Two grams of fresh leaf material harvested from Maverick and ATPS overexpressing soybean plants were individually homogenized with 10 ml of 50 mM HEPES-KOH, pH 7.5, 250 mM sorbitol, 50 mM KOAc, 2 mM Mg(OAc)2, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT and protease inhibitor cocktail. The slurry was subjected to multiple centrifugation steps in order to obtain crude chroplastic, endoplasmic reticulum and cytoplasmic protein fractions 57 . Equal amount of proteins (30 μg) representing the different subcellular fractions were resolved by SDS-PAGE followed by western blot analysis using ATPS antibody as described above.
Amino acid analysis. Dry seeds from four independent transgenic events (ATPS1, ATPS2, ATPS3, ATPS4) and a wild-type soybean line (Maverick) were ground to a fine powder and subjected to hydrolyzed amino acid analysis (Proteomics & Metabolomics Facility at the Center for Biotechnology/ University of Nebraska -Lincoln). Amino acids were quantified using the Waters AccQ-Tag Ultra Kit on an Acquity UPLC system. In this procedure, Cys and Met are quantified as CyA (cysteic acid) and MetS0 2 (methionine sulfoxide), Gln and Asn are converted to Glu and Asp, respectively while Trp is destroyed during hydrolysis. Three biological replicates were used for amino acid measurements. Statistical analysis was performed using JMP 14.0.0 statistical software. Mean amino acid (% mole) were compared via ANOVA and for tests which showed significant differences between wild-type and transgenic seeds means were compared through Tukey's HSD; genotypes which are not significantly different are indicated by overlapping letters.