Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-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 ß-subunit of ß-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.

Calcium regulates the production of nodulation outer proteins (Nops) and precludes pili formation by Sinorhizobium fredii USDA257, a soybean symbiont.

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean cultivars such as 'Peking' but is unable to establish efficient symbiosis with North American cultivars. USDA257 when grown in presence of genistein, a potent nodD-inducing isoflavonoid, secretes at least six nodulation outer proteins (NopX, NopB, NopL, NopP, NopA and NopC) to the extracellular milieu through a type III secretion system. These proteins regulate legume nodulation in a host-specific manner. Here, it is demonstrated that calcium prevents the accumulation of NopB and NopA, and drastically reduces that of NopX and NopL. The inhibitory effect on Nops accumulation appears to be mediated specifically by calcium since other divalent cations such as Mg(2+) and Mn(2+) had no detectable effect. Calcium does not appear to interfere with the secretion of these proteins since Western blot analysis revealed that these Nops do not accumulate inside the cell. The inhibitory effect of calcium on Nops production is mediated at the posttranscriptional level. Studies by the authors indicate that the production of Nops, which function as determinants of host-range, is regulated by calcium.

Sinorhizobium fredii USDA257 releases a 22-kDa outer membrane protein (Omp22) to the extracellular milieu when grown in calcium-limiting conditions.

Calcium, which regulates a wide variety of cellular functions, plays an important role in Rhizobium-legume interactions. We investigated the effect of calcium on surface appendages of Sinorhizobium fredii USDA257. Cold-field emission scanning electron microscopy observation of USDA257 grown in calcium-limiting conditions revealed cells with unusual shape and size. Transmission electron microscopy observation revealed intact flagella were present only when USDA257 cells were grown in calcium-sufficient conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of flagellar preparations from USDA257 cells grown in calcium-limiting conditions showed the presence of a 22-kDa protein that was absent from cells grown in calcium-sufficient conditions. We have cloned and determined the nucleotide sequence of the gene encoding the 22-kDa protein. After successful expression in Escherichia coli, polyclonal antibodies were raised against the recombinant 22-kDa protein (Omp22). Subcellular fractionation analysis demonstrated that Omp22 was predominantly present in the extracellular fraction. Western blot analysis revealed the presence of immunologically related proteins from diverse rhizobia. Immunocytochemical localization of thin sections of USDA257 cells showed specific labeling of protein A-gold particles on protein inclusions found proximal to the cells. Accumulation of Omp22 was greatly reduced when USDA257 cells were grown in the presence of increasing calcium. Northern blot analysis indicated that calcium was the only divalent cation among those tested that down-regulated omp22 expression. An omp22 mutant was able to grow in calcium-limiting conditions at a rate similar to that of wild-type USDA257. Significantly more nodules were initiated by the omp22 mutant than by the wild-type on soybean cultivar Peking grown in calcium-limiting conditions.

Citrate synthase mutants of Sinorhizobium fredii USDA257 form ineffective nodules with aberrant ultrastructure.

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.