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

Abstract

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.

Applications of Genomic Tools in Plant Breeding: Crop Biofortification

“…Increased content of different phenylpropanoids [23] Strawberry Overexpression of a GDP-l-galactose phosphorylase (GGP) gene from Actinidia chinensis under the control of the 35S promoter A 2-fold higher content in ascorbic acid in fruits [200] Banana Expression of a Fe'i banana-derived phytoene synthase (MtPsy2a) gene under the maize polyubiquitin promoter Enhanced β-carotene content in fruit [24] Soybean Overexpression of the bacterial genes crtB (for phytoene synthase) and crtW and bkt1 (ketolase genes) under the control of seed-specific promoters Enhanced accumulation of ketocarotenoids in seeds [201] Overexpression of adenosine 5'-phosphosulfate sulfurylase 1 Higher amounts of sulfate, cysteine, and some sulfur-containing secondary metabolites in seeds [34] Overexpression of a GmDGAT2A gene driven by a seed-specific promoter of Gmole1…”

“…or both [29], are the most common nutritional targets for biofortification strategies, though the improvement in fatty acid composition [30][31][32][33] and the increase in essential amino acids [34][35][36][37] and antioxidants [38][39][40][41] have also been recently included as aims of biofortification programs. This strategy carries multiple advantages.…”

“…Biofortification, i.e., the development of food crops with a high nutritional value per se through both conventional breeding and modern biotechnology techniques, could help in preventing hidden hunger. Micronutrients, minerals [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ], vitamins [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ], or both [ 29 ,], are the most common nutritional targets for biofortification strategies, though the improvement in fatty acid composition [ 30 , 31 , 32 , 33 ] and the increase in essential amino acids [ 34 , 35 , 36 , 37 ] and antioxidants [ 38 , 39 , 40 , 41 ] have also been recently included as aims of biofortification programs. This strategy carries multiple advantages.…”

Progress in soybean functional genomics over the past decade

“…It was found that MGL, a putative methionine γ‐lyase, may be responsible for the accumulation of S‐methylmethionine in soybean seed (Teshima et al ., 2020). Overexpression of the cytosolic isoform of O‐acetylserine sulfhydrylase (OASS) and the plastid ATP sulfurylase isoform 1 improved the cysteine‐rich proteins and sulphur amino acid content in transgenic soybean, independently (Kim et al ., 2012b; Kim et al ., 2020). Rab5a , a small GTPase‐encoding gene, was reported to be involved in post‐Golgi trafficking of storage proteins in developing soybean cotyledons (Wei et al ., 2020b).…”

Quantitative proteomic analyses reveal the dynamics of protein and amino acid accumulation during soybean seed development

“…While soybean is considered a valuable commodity of vegetable protein and oil supply for human food and animal feed, it has a limited amount of sulfur-containing amino acids such as methionine and cysteine. Therefore, several strategies, including metabolic engineering, have been undertaken to enhance the sulfur-containing amino acids in soybean seed [26,27].…”