Plant Science Division, University of Missouri, Columbia, MO, 65211, USA.
Plant Genetics Research, USDA-Agricultural Research Service, University of Missouri, 108 Curtis Hall, Columbia, MO, 65211, USA.
Sci Rep. 2020 Sep 14;10(1):14989. doi: 10.1038/s41598-020-72134-z.
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.
三磷酸腺苷硫酸化酶是一种将硫酸盐转化为腺苷 5′-磷酸硫酸（APS）的酶，在植物的硫代谢中起着重要作用。在这项研究中，我们在不含有其转运肽的情况下，在 35S CaMV 启动子的控制下，在转基因大豆中表达了大豆质体三磷酸腺苷硫酸化酶同工酶 1。亚细胞分级和免疫印迹分析表明，ATP 硫酸化酶同工酶 1主要在细胞质中表达。与未转化的植物相比，发育种子中的 ATP 硫酸化酶活性约高 2.5 倍。高分辨率 2-DE 电泳和免疫印迹分析表明，过表达 ATP 硫酸化酶的转基因大豆种子中 β-伴大豆球蛋白的β-亚基积累水平非常低。相比之下，与非转基因野生型种子相比，转基因大豆植株中富含半胱氨酸的 Bowman-Birk 蛋白酶抑制剂的积累量高出数倍。与野生型种子相比，转基因种子的总蛋白质含量降低了约 3%。通过 LC-MS 和 GC-MS 对代谢物进行分析，共定量了 124 种种子代谢物，其中 84 种在 ATP 硫酸化酶过表达种子中的含量较高，40 种在 ATP 硫酸化酶过表达种子中的含量较低。与野生型种子相比，硫酸盐、半胱氨酸和一些含硫次生代谢物在 ATP 硫酸化酶过表达种子中积累量更高。此外，ATP 硫酸化酶过表达种子中还含有显著更高水平的磷脂、溶血磷脂、二酰基甘油、甾醇和硫脂。重要的是，ATP 硫酸化酶的过表达导致转基因种子中蛋白质结合的半胱氨酸和蛋氨酸含量分别增加了 37-52%和 15-19%。我们的结果表明，操纵关键硫同化酶的表达水平可以用来提高大豆种子的营养价值。
