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Dep. of Agronomy and Zoology/Genetics, Iowa State Univ., Ames, IA 50011-1010
Corresponding author (jimsande{at}iastate.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AOAC, Association of Official Analytical Chemists • EMS, ethyl methanesulfonate • ICP-AES, inductively coupled plasma atomic emission spectroscopy • OAS, O-acetylserine
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Soybean seed storage proteins are composed primarily of two general classes, glycinins and ß-conglycinins. The glycinins, or 11S proteins, constitute approximately 40% of total seed protein and are encoded by a gene family (Nielsen et al., 1989). The ß-conglycinins, also encoded by a gene family, are smaller proteins (7S) and constitute approximately 30% of soybean seed protein (Coates et al., 1985; Harada et al., 1989). Whereas the glycinins contain a standard methionine and cysteine content (Nielsen et al., 1989), the ß-subunits of ß-conglycinin lack methionine and contain either one or no cysteine residues (Coates et al., 1985; Harada et al., 1989). Although soybean seeds contain low molecular weight proteins that are either cysteine-rich or methionine-rich (Biermann et al., 1998; George and de Lumen, 1991), the degree of methionine and cysteine deficiency in soybean protein is generally determined by the relative abundance of the ß-subunit of ß-conglycinin (Coates et al., 1985; Paek et al., 1997).
Both in vitro and in planta, high levels of methionine inhibit synthesis of the ß-subunit of ß-conglycinin (Gayler and Sykes, 1985; Holowach et al., 1984; Naito et al., 1994). Conversely, soybean plants provided adequate or excess reduced nitrogen during pod filling preferentially synthesize the ß-subunit because of an apparent methionine deficiency during late pod filling (Imsande, 1998; Imsande and Schmidt, 1998; Paek et al., 1997). Hence, if the methionine concentration within soybean pods and/or seeds were increased during late pod filling, when the ß-subunit of ß-conglycinin is being formed de novo (Harada et al., 1989), synthesis of the ß-subunit would be inhibited. Therefore, the relative abundance of methionine and cysteine in soybean seed storage proteins would be increased (Holowach et al., 1984).
The methionine biosynthetic pathway in plants, including soybean, has been reviewed (Hughes et al., 1999; Kim and Leustek, 2000; Ravanel et al., 1998). At least two separate reactions contribute to the regulation of methionine biosynthesis. First, the half-life of the mRNA encoding cystathionine 
-synthase, which catalyzes the condensation of cysteine and O-phosphohomoserine producing cystathionine, is reported to be shortened by elevated concentrations of methionine or a methionine metabolite (Chiba et al., 1999). Thus, the internal concentration of methionine may regulate the biosynthesis of the enzyme that catalyzes the first committed step in methionine biosynthesis. On the other hand, cysteine, a reactant in methionine biosynthesis, is formed from O-acetylserine (OAS) and sulfide. Cysteine biosynthesis is catalyzed by a bifunctional complex composed of two molecules of homodimeric O-acetyl-serine sulfhydrase and one molecule of homotetrameric serine acetyl-transferase (Droux et al., 1998). The activity of this complex, and hence the rate of cysteine biosynthesis, may be controlled by the intracellular concentration of OAS (Kim et al., 1999). Because cysteine is a reactant in methionine biosynthesis, the internal concentration of cysteine may contribute to the regulation methionine biosynthesis (Droux et al., 1998; Kim et al., 1999; Saito et al., 1995). Indeed, it has been reported that the internal concentration of OAS may regulate the relative abundance of the ß-subunit of ß-conglycinin (Kim et al., 1999). Thus, a strategically located mutation in the genes encoding either cystathionine -synthase, serine acetyl-transferase, or O-acetyl-serine sulfhydrase could promote methionine over-production and the genetic improvement of sulfur amino acid-deficient soybean protein. Also, competition for O-phosphohomoserine, a reactant in the synthesis of both methionine and threonine, may restrict methionine biosynthesis (Curien et al., 1996; Kim and Leustek, 2000; Ravanel et al., 1998).
Ethionine, a chemical analogue of methionine, is highly toxic to most organisms (Alix, 1982). Because of their similarity in chemical structure and composition, an increased concentration of methionine lessens the toxicity of low concentrations of ethionine. Thus, low concentrations of ethionine have been used successfully to select and identify methionine over-producing variants, including soybean tissue culture lines (Gonzales et al., 1984; Inaba et al., 1994; Langille et al., 1998; Madison and Thompson, 1988). All 26 of the ethionine-resistant soybean tissue culture lines isolated contained an elevated internal methionine concentration, the mean of which, 41.7 nmol g-1 fresh weight of callus, was 8.7 fold greater than that of the parental line (Madison and Thompson, 1988). Also, all 26 lines contained an elevated internal concentration of S-methylmethionine, the mean of which, 43.3 nmol g-1 fresh weight of callus, was 11.7 fold greater than that of the parental line. S-methylmethionine, which has been found in many plant species, is thought to serve as a methionine reserve (Mudd and Datko, 1990). Ethionine resistance also has been used to isolate methionine over-producing Arabidopsis thaliana (L.) Heynh. plants (Inaba et al., 1994).
During soybean pod filling, leaf proteins are hydrolyzed and the salvaged nitrogen is translocated efficiently by the phloem to the developing pods and seeds primarily as asparagine and glutamine (Pate et al., 1984; Streeter, 1979). Because of the reactivity of the sulfhydryl group of cysteine, little or no cysteine is transported in the phloem. Rather, soybean, as well as other plants, transports reduced sulfur in the phloem primarily as S-methylmethionine and glutathione (Bourgis et al., 1999). Although developing soybean pods and seeds are capable of reducing sulfate (Sexton and Shibles, 1999), the amount of methionine obtained from seed-reduced sulfate and translocated S-methylmethionine is inadequate to repress synthesis of the ß-subunit during late pod filling (Imsande and Schmidt, 1998; Naito et al., 1994; Paek et al., 1997). Hence, to increase the relative abundance of the sulfur amino acids in soybean protein, the plant must maintain a high concentration of methionine at the time and site of seed storage protein synthesis so that synthesis of the ß-subunit will be repressed.
The objectives of these studies were to mutagenize soybean seeds, to develop a screening procedure for the selection of methionine over-producing mutant lines, and to isolate genotypes whose seeds were enriched in organic seed sulfur.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phenotype of Methionine Over-Production
To determine the phenotype of methionine over-production in vegetative tissues, soybean plants were grown hydroponically in the glasshouse in either a standard hydroponic growth medium (Imsande and Ralston, 1981; Ralston and Imsande, 1983; Imsande, 1998) or the standard medium supplemented with 0.05, 0.10, or 0.20 mM methionine.
Mutagenesis and M1 Seed Production
Approximately 120 000 Kenwood 94 seeds were washed with water and soaked approximately 14 h with aeration (May 21, 1995). Each of the three aeration barrels contained approximately 40 000 seeds. At 0600 h, each barrel was drained, the seeds quickly rinsed with water, and 24 L of 0.5% (v/v) ethyl methanesulfonate (EMS) in 0.1 M KH2PO4 buffer, pH 6.0, were added to each barrel (Carroll et al., 1986). After a 6-h treatment with aeration, the liquid in each barrel was drained into a safety container and the seeds were washed with water for 1 h. The mutagenized seeds (M0) were immediately hand-planted in prepared rows in the field. At maturity, the M0 plants were threshed and the M1 seeds bulked.
Phenotypic Selection of Field-Grown Plants
Approximately 91 500 bulk-harvested M1 seeds, 26 per meter, were machine-planted (25 May 1996). From the R1 through the R6 stages (Fehr and Caviness, 1977), the approximately 65 000 viable plants were screened visually each week. Plants with dark-green leaves were identified. If the dark-green phenotype continued throughout R6, the identified plants were tagged and single-plant threshed. A random sample of 10 M2 seeds from each of the 82 plants was assayed by S-analysis for seed sulfur concentration. Remnant M2 seeds from the 10 plants whose seeds showed a significantly high seed sulfur concentration were grown at the University of Puerto Rico, Iowa State University Soybean Nursery, Isabela Substation, Isabela, Puerto Rico, (winter-spring 1997) for generation advance. The M3 seeds from each of these 10 lines were collected and assayed for sulfur concentration, and the remnant seeds were field-grown in Ames, IA, (summer 1997) yielding M4 seeds.
Selection for Ethionine Resistance
Plants produced by the M1 seeds were screened for methionine over-production as follows.
Step 1
Commercial germination paper (Anchor Paper, Hudson, WI) was halved longitudinally, washed with deionized water, and autoclaved. By sterile technique, 12 M1 seeds were equally spaced between two sheets, and the sheets were rolled to produce a column of 12.5 cm. A rubber band was placed immediately below the seeds. Seven rolls were placed vertically in a sterile cup 10 cm in diameter containing 250 mL of aqueous ethionine (0.75 mM), and the rolls and the upper portion of the cup were enclosed with a large plastic bag. A rubber band was used to hold the plastic bag firmly to the side of the cup. The bags were inflated with air to provide CO2 and space for seedling growth. Capillary action carried the ethionine solution up the rolls toward the seeds. As the seeds germinated, geotropism directed the roots downward. However, as the roots approached the ethionine solution, root growth was increasingly inhibited. After a 7-d treatment, generally less than 1% of the seeds exposed to 0.75 mM ethionine produced roots longer than 11 cm and hence entered the solution, whereas 75% of the control seeds, exposed only to water, produced roots longer than 11 cm. From each of eight experimental cups, the three seedlings with the longest, whitest, and most healthy appearing roots were selected daily for Step 2. Thus, 672 seeds typically produced 24 seedlings for Step 2 of the screening procedure.
Step 2
Selected 7-d-old seedlings were grown hydroponically in a lighted plant growth room (Imsande and Ralston, 1981; Imsande, 1998; Imsande and Edwards, 1988). After 21 d, relatively large plants with noticeably dark green leaves, usually two or three of the original 24, were transferred to the glasshouse for continued hydroponic growth. All media were renewed twice weekly.
Step 3
Plants were grown hydroponically in the glasshouse until they reached maturity. During this period, the phenotype of each plant was noted weekly. Those plants with dark green curly leaves and unusually large bulging pods were selected for progeny testing. Pods were hand harvested, shelled, and the number of seeds noted. Seeds (M2) were subsequently weighed and planted in the field for progeny testing. Of the approximately 50 500 plants screened by this procedure, 240 plants with a putative high sulfur phenotype were selected. A random sample of 10 M2 seeds from each of these plants was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis for seed sulfur content. Remnant M2 seeds from the12 plants whose seeds showed a significantly higher seed sulfur content were field-grown during the summer of 1996 for generation advance. The M3 seeds from each of these 12 lines were collected, assayed for sulfur content, and remnant seeds planted at the Puerto Rico nursery to produce M4 seeds.
Genetic Crosses
On 30 May 1997, 60 M3 seeds from each of seven high seed-sulfur lines obtained by field phenotype selection and 60 M4 seeds from each of 10 lines obtained by ethionine screening were field planted at Hinds Farm, Ames, IA, for crossing. Reciprocal crosses were performed among each pair of the 17 lines with all lines as both female and male. F1 seeds were potted in October 1997 and F1 plants were grown in the glasshouse. Plants were threshed individually at maturity (March-April 1998).
Seed Sulfur and Nitrogen Determinations
On 21 May 1998, 60 F2 seeds from each selected line, 60 M5 seeds from each selected line and 60 control seeds were hand planted in adjacent 3-m rows at Hinds Farm. On 19 May 1999, 60 F3 seeds from each selected line, 60 M6 seeds from each selected line, and 60 control seeds were hand planted in adjacent 3-m rows at Hinds Farm.
Harvested seeds were dried at 40°C, to approximately 130 g kg-1 moisture, for seed yield analysis. Subsequently 10 medium-sized seeds from each M3 plant were selected at random, dried at 60°C for 24 h, ground in a coffee bean grinder and again dried at 60°C for 24 h. This procedure removed most of the residual seed moisture. Duplicate 100-mg samples were wetted with 2.5 mL of a HNO3 and H2O2 solution (Finch et al., 1990; Imsande and Schmidt, 1998; Sah and Miller, 1992; Soon et al., 1996), digested with a microwave system (Model MDS 2000, CEM Corporation, Matthews, NC), and submitted to ICP-AES analysis (Pilon et al., 1990; Imsande and Schmidt, 1998). Commercial wheat (Triticum aestivum L.) preparations were used as a sulfur standard. Seed nitrogen concentrations were determined by micro-Kjeldahl analysis on duplicate samples, approximately 65 mg each, of the dried seeds. Where indicated, variations in seed sulfur percentage and mmol g -1, in seed nitrogen percentage and mmol g-1, and in the S/N molar ratios were analyzed statistically (ANOVA).
Amino Acid Composition
Amino acid compositions of finely ground soybean seeds were performed by the Iowa State University Protein Facility using a 420A Derivatizer/120A Analyzer, Applied Biosystems, Inc, according to the AOAC procedure (1999). The Protein Facility states that the error in amino acid mole percentages is less than 5%. Plants described in Table 3 were the seed source. Seed coats were removed prior to seed grinding. Ten F4 seeds were selected at random from a single H82 x 10a1 plant, from each of two H82 x 20a2 plants and from a single Kenwood 94 control plant. Cysteine content was determined as cysteic acid, with lysozyme and cysteic acid as standards, according to Spencer and Wold (1969).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M1 seeds (50 568) were germinated in the presence of 0.75 mM aqueous ethionine and screened for ethionine resistance by relative root length, dark green leaf coloration, and plant growth rate (see Methods). By means of this procedure, 240 M1 plants were obtained. At maturity, M2 seeds from the 40 most promising plants selected by ethionine resistance were tested for seed sulfur concentration, and remnant seeds were field-planted (1997). The M3 seeds were tested for seed sulfur concentration and seeds from the 10 promising M3 high-seed-sulfur lines were advanced to M4 seeds. Seeds (M4) of nine of the 240 lines obtained by the original ethionine selection procedure contained an increased seed sulfur concentration (Table 1).
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Genetic Crosses
To obtain the 18 lines presented in Table 1, more than 115 000 plants were screened. Because it is statistically unlikely that all 17 lines resulted from a single mutational event, reciprocal crosses were performed between every pair of the 17 lines. For F3 seeds, the mean seed-nitrogen concentration, the mean seed-sulfur concentration, and the extremes in seed-sulfur concentrations of each line are shown in Table 2. Also, values for two of the parental lines used in crossing, H52 and H82, one high seed-nitrogen line, 131-9b, and the experimental control, Kenwood 94, are presented. Seed-sulfur concentrations in most of the lines presented in Table 2 are significantly greater than that of the parental control, Kenwood 94.
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Amino Acid Composition of Soybean Seeds
To determine whether or not the seed-sulfur increase represented an increase in sulfur amino acids, the amino acid composition of a few lines was examined (Table 4). The amino acid mole percentages for methionine and cysteine in line H82 x 20a2 were 1.83% and 1.69%, respectively, whereas the mole percentages for methionine and cysteine in Kenwood 94 control seeds were 1.51% and 1.32%, respectively. These results indicate that the mole percentages for methionine and cysteine in line H82 x 20a2 were approximately 22 and 28% greater, respectively, than those of Kenwood 94 control seeds. Total seed sulfur concentration in the mutant line, however, was only 18% greater, i.e., 0.1184 vs 0.1397 mmoles g-1 (Table 3). Calculations show that approximately 90% of seed sulfur was protein sulfur. Thus, the amino acid mole percentages of methionine and cysteine in H82 x 20a2 were each approximately 20% greater than that of Kenwood 94. These differences are statistically significant. The nitrogen concentration of seeds from line H82 x 20a2, approximately 4.63 mmoles N g-1 seeds, was similar to that of the Kenwood 94 parental line, 4.58 m moles N g-1 seeds (Table 3). Thus, the S/N ratio of H82 x 20a2 seeds was approximately 16.2% greater than that of the parental line.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two procedures for the isolation of methionine over-producing soybean plants were developed. Plants whose seeds contained an elevated sulfur concentration were selected by both the ethionine resistance assay and by the methionine over-producing phenotypic procedure (Tables 1 and 2). Plants with the highest seed sulfur concentration, however, were obtained by genetically crossing high seed-sulfur lines obtained from the two selection procedures (Table 3).
A typical 1-kg feed ration for animals contains 300 g soybean meal, which is approximately 50% protein. Thus, a 1-kg ration contains approximately 150 g of soybean protein. Published amino acid mole percentage values for L-methionine of soybean protein generally range from 1.22 to 1.50 (Kitamura and Kaizuma, 1981; Ogawa et al., 1989) with a mean of 1.40 (George and de Lumen, 1991). Because the molecular weight of methionine, 149.2 g, is significantly greater than the mean molecular weight of the other 19 amino acids, approximately 130 g, a 1-kg ration contains approximately 2.4 g of L-methionine derived from soybean protein [i.e., 1.40% x (149.2 ± 130) x 150 g = 2.4 g]. Also, a typical 1-kg ration is supplemented with 1 g of DL-methionine. Hence, the methionine concentration of soybean protein must be increased approximately 21% to provide as much L-methionine as is present in the supplemented ration, or 42% to equal the DL-methionine supplementation. Our results show that line H82 x 20a2 has a methionine mole percentage of 1.84 (Table 4), which is approximately 31% greater than the 1.40 mole percentage methionine found in a random sample of soybean meal. Because the mole percentage of cysteine in line H82 x 20a2 also is increased approximately 20%, rations prepared fromg the H82 x 20a2 genetic line should not require methionine supplementation.
These high sulfur lines were produced by a standard mutagenic procedure and not by genetic engineering. Protein obtained from these high-sulfur seeds would provide a large financial saving to the livestock industry and also would be beneficial to the many people of the world who use soybean as a major protein source.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication June 13, 2000.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-synthase mRNA stability in Arabidopsis. Science 286:1371–1374.-synthase in soybean. Plant Sci. 146:69–79.-synthase in Arabidopsis thaliana produces partial methionine auxotrophy and developmental abnormalities. Plant Sci. 151:9–18.
' and ß subunit genes in transgenic plants. Plant Molec. Biol. 11:109–123.[ISI]This article has been cited by other articles:
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  H. B. Krishnan Engineering Soybean for Enhanced Sulfur Amino Acid Content Crop Sci., January 31, 2005; 45(2): 454 - 461. [Abstract] [Full Text] [PDF]
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D. Chronis and H. B. Krishnan Sulfur Assimilation in Soybean: Molecular Cloning and Characterization of O-Acetylserine (Thiol) Lyase (Cysteine Synthase) Crop Sci., September 1, 2003; 43(5): 1819 - 1827. [Abstract] [Full Text] [PDF]
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