ABSTRAC1Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental L-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S a-subunit gene. Concomitant with the absence of the a-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of 6-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.Soybean seed proteins lack sufficient quantities ofsulfur amino acids to satisfactorily meet the dietary requirements of some monogastric animals. In this respect, soybeans are typical legume seeds. Soybeans, however, accumulate about twice as much protein in their seeds as other legumes, and up to four times as much protein as grain seeds. The nutritional quality of soybean and other legume seeds could be improved by raising the sulfurcontaining amino acid content of their proteins (37). Since the 7S and Il S storage protein fractions constitute about 70% of total seed protein, they have been the focus of efforts to raise protein methionine content. The soybean 7S fraction consists of three major subunits, the a'-, a-, and a-subunits. (29,31) In vitro experiments were designed so that sets of 5 to 46 cotyledon pairs were subjected to control and experimental treatments. Individual seeds were divided so that one-half was placed in complete medium lacking methionine, and the other half in complete medium containing 8.4 mm L-methionine.Rates of growth and protein accumulation were variable in seeds grown on plants. Seeds matured more slowly in the winter than in the summer. Maturity was therefore measured by increments of seed fresh weight, which was found to be a more consistent measure of the time course of development than days after flowering (13,18
Immature soybean (Glycine max L. Merrill cv Provar) cotyledons were grown aseptically for 6 days in complete culture medium with zero, deficient (17 micromolar), sufficient (1.5 millimolar), or supraoptimal (7.5 millimolar) levels of sulfate. Some cotyledons at each sulfate concentration were supplemented with L-methionine. No sulfate or 17 micromolar sulfate were inadequate for growth and protein accumulation, but all the major subunits of the 7S and 11S storage protein fractions were detected. The ratio of 11S to 7S proteins was <1.0. Addition of 8A millimolar methionine overcame the restriction of cotyledon growth and protein accumulation in the sulfate-deficient media, and the ratio of IIS to 7S proteins was significantly increased. The amino acid compositions of the 7S and 11S fractions from sulfate-sufficient cotyledons and from sulfate-deficient cotyledons were not significantly different. There was no difference in fresh weight or total protein accumulation in cotyledons grown in 1.5 millimolar or 7.5 millimolar sulfate. At 7.5 millimolars sulfate, the IIS to 7S ratio was significantly increased, and the amount of ti-subunit in the 7S fraction decreased. At all sulfate levels supplemented with methionine, the 11S to 7S ratio was greater than 1, and no ,B-subunit was detected in the 7S fraction. Supplemental methionine in media of any sulfate concentration increased growth and protein methionine content to a greater extent than high (7.5 millimolar) sulfate only. Adding supraoptimal sulfate is not equivalent to supplementing with methioaine. Results of this study of in vitro growth are compared to results of studies of seed development on intact plants supplied with various sulfur concentrations.
In previous studies (GL Creason et al 1983 Biochem Biophys Res Commun 117: 658-662; LP Holowach et al. 1984 Plant Physiol 74:576-583), we have shown that when soybean (Glycine max L. Merril cv Provar) cotyledons are cultured in medium supplemented with i-nmethionine, the ti-subunit of 7S protein and ft-mRNA are absent. We have carried out further studies on the mechanism of the methionine action.In one experiment, cotyledons were cultured for 16 days with or without methionine. After 4 days, some cotyledons were trnsferred from methionine-supplemented to basal (no methionine) medium and vice versa. In basal medium, ti-subunit was detected at 4 days whereas in methioninesupplemented medium, no t-subunit was present. When cotyledons were transferred from basal to methionine-supplemented medium, the t-subunit increased within a 4 day period and then remained constant (on a per cotyledon basis). This result indicated that methionine was not acting by accelerating the degradation of the i-subunit. Four days after tnmfer from supplemented to basal medium cotyledons contained ti-subunit, thus demonstrating that the inhibition was reversible. During this time, the uncombined methionine declined from 7 to 1.5 gnmoles methioniae per gram fresh weight. When ,6-mRNA was measured-by in vitro translation, functional 6-mRNA was absent in tissue that was not accumulatingsubunit. The messenger RNA for the t-subunit had a half-life of about 1 day in the presence of methionine. Hybridization of cotyledon mRNA with cDNA complementary to -mRNA revealed that the 1700 nucleotide ti-mRNA was not present in supplemented cotyledons. Thus, expression of the ti-subunit gene is controlled at the level of rnswription, RNA processing, or RNA turnover, rather than at the level of translation. The 7S fraction of soybean comprises three major subunits: a, a ', and ,B (27). Each is detectable in seeds developing on plants, and in immature cotyledons cultured in vitro (12). However, when the basal culture medium is supplemented with L-methionine, the l-subunit is not detectable (7,12). The f-subunit contains no methionine (12,27). The behavior of the ,B-subunit gene is unique in supplemented cotyledons because it is the only major storage protein gene whose expression is suppressed by methionine (12). The absence of f-subunit in methionine-supplemented cotyledons is correlated with the absence offunctional f-subunit mRNA, assayed by in vitro translation (7).Our goals in this paper were: (a) to determine ifthe methionine inhibition of A-subunit synthesis was reversible, (b) to determine if the fl-subunit was stable in the presence of methionine, (c) to determine the rate ofchange in the concentration ofuncombined methionine after transfer from methionine-supplemented to basal medium, (d) to measure the rate of degradation of fl-mRNA in the presence of methionine, and (e) to use Northern hybridization to determine ifsequences offl-mRNA were present under conditions where there was no functional fl-mRNA detecte. MATERIALS AND METHODSDesign ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.