The Relationship Between Sulfur Metabolism And Fuctional Protein Components In Soybean (Glycine Max (L.) Merrill)

This research was conducted at the experiment station of Northeast Agricultural University from 2007 to 2008. Two cultivars, Dongnong46(high-oil), Heinong35 (high-protein) were tested in this research by means of pots experiments with three sulfur fertilizer application levels(0 g·kg -1soil,0.2 g·kg -1 soil,0.8 g·kg -1 soil)and two nitrogen fertilizer application(50 kg(N)·hm-2,100(N) kg·hm-2)to study the co-operative effects of sulfur and nitrogen fertilizer on sulfur and nitrogen concentration of vegetative organs, metabolism of key enzymes and the quality of soybean. The results showed that:The sulfur and nitrogen concentration of vegetative organ were influenced by co-operative effects of sulfur and nitrogen fertilizer. The sulfur and nitrogen concentration in leaves, stems, petioles (except the concentration of nitrogen) and pods showed a declining tendency during the growth period, while that in the seeds showed an increased tendency. The application of sulfur fertilizer raised the sulfur concentration of vegetative organ and the main effect of sulfur fertilizer was bigger than that of nitrogen fertilizer. The main effect of nitrogen application fertilizer was significant in stems and seeds, while inconspicuous in leaves and pod shells. The application of sulfur can promote the absorption of nitrogen in leaves while enough sulfur supply. The ratio of N/S was very wide and ranged from 1.52 to 20.26. The N/S of leaves, pods shell and seeds were stable, and raised significantly when there were no enough sulfur fertilizer.The content of amino acids and GSH were significantly influenced by co-operative effects of sulfur and nitrogen fertilizer during the seed filling-period. The application of sulfur and nitrogen fertilizer raised the content of Cys, Met and total sulfur containing amino acids which showed an increase tendency during the stage of seeds filling. The treatment of CK2 raise the content of principal amino acids like Asp and Glu significantly at the early stages of seed development, and the difference between application disappeared accompany with the filling of seeds. The correlation between N/S and part of the principal amino acids were positive, and the correlation between N/S and sulfur containing amino acids was negative. The effects of application of sulfur on GSH were regulated by growth stages. The content of GSH in leaves showed no significant difference before the seed filling-period and keep a small cloth declining tendency to the filling period,and from that on ,the treatment without sulfur fertilizer declined dramatically, and the treatment with application of sulfur keep stable until the mature of seeds. There was in-conspicuous effect on GSH content under nitrogen application.A full-length cDNA clone encoding ATPS was obtained by RT-PCR. The sequence covers 1310 nucleotide. The length of ORF covers 801bp, begins at the position of 306 and ends at the position of 1106 of the nucleotides sequence encoding a 29.93 -kDa protein of 267 amino acids. The theoretical isoelectric point of the protein was estimated to be 6.20. The nucleotides sequence of ATPS has a close genetic relationship with leguminous plants. The ATPS amino acids sequence contains an nt_trans (nucleotidyl transferase) superfamily and the active site around at the 150th of the amino acids sequences. The relatively expression amount of ATPS mRNA was regulated by development mechanism. It showed a declining tendency during the seeds filling period. There were significant differences at the early stage of seed development and than disappeared later. Both the deficiency and excessive of sulfur feed back the expression of ATPS. The application of nitrogen can promote the expression of ATPS and the differences disappeared at the final stages of development.A full-length cDNA clone encoding APR was obtained by RT-PCR. The sequence covers 1506 nucleotide. The length of ORF covers 1026 bp, begins at the position of 323 and ends at the position of 1348 of the nucleotides sequence encoding a 38.09 -kDa protein of 342 amino acids. The theoretical isoelectric point of the protein was estimated to be 6.35. The APR amino acids sequence contains a PAPS_ reductase and Thioredoxin (TRX)-like superfamily conserved domains and the active site between 100 to 150 amino acids of the amino acids sequence. The relatively expression amount of APR mRNA was regulated by development mechanism. It showed a declining tendency during the seeds filling period. It was promoted by lack of sulfur supply. HN35 showed irregular responsion to sulfur and nitrogen treatments.A full-length cDNA clone encoding SAT was obtained by RT-PCR. The sequence covers 911 nucleotides. The length of ORF covers 620bp, begins at the position of 184 and ends at the position of804 of the nucleotides sequence encoding a 21.68 -kDa protein of 207 amino acids. The theoretical isoelectric point of the protein was estimated to be 7.78. The nucleotides sequence of SAT has a close genetic relationship with leguminous plants. The SAT amino acids sequence contains cysE, LbetaH superfamily and SATase_N superfamily conserved domains. The relatively expression amount of SAT mRNA showed high level during the growth stage. There were significant differences at the early stage of seed development and than disappeared at the 28th of seed filling-period. The relatively expression amount of SAT mRNA was inhibited by sulfur deficiency and promoted by sulfur application, and the excessive sulfur supply decline the expression level.A full-length cDNA clone encoding OAS-TL was obtained by RT-PCR. The sequence covers 1059 nucleotide. The length of ORF covers 854bp, begins at the position of 58 and ends at the position of 912 of the nucleotides sequence encoding a 28.49 -kDa protein of 284 amino acids. The theoretical isoelectric point of the protein was estimated to be 5.45. The nucleotides sequence of OAS-TL has a close genetic relationship with other species. The activity of OAS-TL declined during the seed filling-period, both the deficiency and excessive of sulfur inhibited the activity of OAS-TL, the activity of OAS-TL was higher in the treatment of N100 than N50, but the differences of sulfur and nitrogen applications disappeared at the final stages of development. The relatively expression amount of OAS-TL mRNA was regulated by development mechanism. The expression level was higher at the early stage and than declined along with the maturation of seeds. A suitable sulfur accompany nitrogen application can keep the expression level on a high status. The expression of OAS-TL mRNA was higher N50S20 and N100S80.Co-operative effects of sulfur and nitrogen influence the quality traits of soybean. The application sulfur at N100 level raised the content of protein of DN46. The protein content of HN35 was increased by the application of sulfur. The content of total amino acids, essential amino acid and sulfur containing amino acids of DN46 were lower than those of HN35. The content of sulfur containing amino acids of DN46 and HN35 were higher at N100 than that at N50, and higher under applications with sulfur than the application without sulfur. The application of sulfur and nitrogen declined the oil content. The total content of fatty acids increased under sulfur application with N50.The application of nitrogen declined the content of oleic acid and linoleic acid, while increased the content of linolenic acid.