Occurrence of mRNA for storage protein in dry soybean seeds

Poly(A)-containing RNA has been isolated from the cotyledons of soybean seeds by adsorption on a poly(U)-Sepharose column. Approximately 0.15% of the total soybean RNA applied bound to the column. The bound RNA (poly(A)-containing RNA) was shown to be mRNA by its ability to serve as template in a cell-free system derived from wheat germ. Poly(A)-containing RNA was polydisperse, migrating from approximately 50,000 to 700,000 daltons with a mean of 150,000 daltons in polyacrylamide gel electrophoresis. The size of the poly(A) portion of this RNA was in the range of 55 to 290 nucleotides. The adenylic acid content of the presumed poly(A) fragment was about 95%. The radioactive products of translation directed by the poly(A)-containing RNA in the wheat germ cell-free system were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation using antisera against beta-conglycinin and glycinin. The results of this investigation show that mRNAs for the subunit proteins of the major components of a soybean storage protein exist in the poly(A)-containing RNA preparation obtained from the cotyledons of dry soybean seeds.     

Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro

Supplemental 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 beta-subunit gene. Concomitant with the absence of the beta-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 beta-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.     

Differential tissue-specific response to sulfate and methionine of a soybean seed storage protein promoter region in transgenic Arabidopsis

Expression of the gene encoding the beta subunit of beta-conglycinin, a major soybean seed storage protein, is upregulated by sulfur deficiency and downregulated by methionine (Met). The tissue-specificity of these regulatory mechanisms was studied using a sulfate-responsive region (beta(SR)) from the beta subunit gene promoter. Transgenic Arabidopsis thaliana lines were generated carrying a green fluorescent protein (GFP) reporter gene under control of the cauliflower mosaic virus 35S RNA promoter with a tandem repeat of the beta(SR) element, referred to as the P35S::beta(SR)x3: GFP transgene. Upregulation of P35S::beta(SR)x3:GFP by sulfur deficiency was strongest in leaf margins, where symptoms of sulfur deficiency first appear. P35S::beta(SR)x3:GFP was also upregulated at 2 d after a medium shift from sulfur-sufficient to sulfur-deficient conditions, suggesting that the chimeric promoter is an efficient indicator of sulfur nutritional status. Analysis of transgene expression in a Met-overaccumulating mto1-1 mutation background revealed that the beta(SR) region carries sufficient information for downregulation of promoter activity by Met in developing seeds, but not in young rosettes. Comparisons with another transgenic line, in which the full-length beta promoter is active in non-seed tissues, also suggested that at least two separate tissue-specific mechanisms exist for the downregulation of the beta promoter by Met.     

Influence of dietary methionine level on the liver metallothionein mRNA level in rats

The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Effect of methionine on growth and protein composition of cultured soybean cotyledons

lmmature soybean cotyledons were cultured in vitro on a ‘complete’ medium with and without supplementation with methionine. The supplement increased dry wt by 23 %. The growth increase indicated that under these conditions the cotyledons could not synthesize methionine rapidly enough to supply the methionine required for maximum protein synthesis. This indication was supported by finding that aminoacylation of methionyl-transfer RNA was increased 18 % by methionine supplementation. Supplemental methionine also increased the methionine content of the protein fraction by more than 20 %, decreased the arginine content by 11 % and significantly affected several other amino acids. These latter results indicate that the amino acid composition of seed protein can be influenced by the supply of amino acids.     

Glycinin A5A4B3 mRNA: cDNA cloning and nucleotide sequencing of a splitting storage protein subunit of soybean

The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5'-terminal non-translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3'-terminal non-translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full-sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post-translational processing to generate an intermediary subunit complex (A5-B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non-covalent interactions. From the results obtained by the Chou-Fasman rules we speculated that the two post-translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.     

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from -carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an -helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core -barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

Adjustments of the protein level for slaughter pigs and supplementation with lysine and methionine

In two replicate experiments, pigs were fed on a diet in which protein was progressively reduced as they grew from 20 to 70 kg, and thereafter on a cereal mixture alone (treatment VPC), or on a diet in which the protein content was the same from beginning to slaughter, at 95 kg live weight (treatment FPC).Diet VPC resulted in statistically faster growth and better feed efficiency than FPC. Diet VPC, with 0.12% lysine added to the cereal component (VPCL), significantly improved the growth rate in comparison to both VPC and FPC, and also feed efficiency as compared to FPC. Methionine added at a level of 0.1% instead of lysine (VPCM) did not produce any improvement.Carcass quality was not significantly affected by the various treatments.Compensatory growth was not visible on FPC, which was deficient in protein in the beginning and excessive later.     

Cloning of mRNA sequence coding for sex-specific storage protein of Bombyx mori

A major plasma protein, referred to as SP 1, exhibits sex- and stage-specific expression during the larval development of the silkworm, Bombyx mori. We have isolated a plasmid clone bearing a part of mRNA sequence coding for SP 1 and quantitated the amount of SP 1 mRNA by means of RNA blot hybridization using the cDNA probe. The developmental change in the amount of SP 1 mRNA in the fat body closely reflected that of the hemolymph concentration of SP 1, indicating that the biosynthesis of SP 1 is regulated in a sex- and developmental stage-specific fashion at the level of mRNA.     

Nitrogen and methyl jasmonate induction of soybean vegetative storage protein genes

Vegetative storage protein (VSP) and VSP mRNA levels in soybean (Glycine max) leaves correlated with the amount of NH₄NO₃ provided to nonnodulated plants. The mRNA level declined as leaves matured, but high levels of N delayed the decline. This is consistent with the proposed role for VSP in the temporary storage of N. Wounding, petiole girdling, and treatment with methyljasmonate (MeJA) increased VSP mRNA in leaves 24 hours after treatment. The magnitude of the response depended on leaf age and N availability. N deficiency essentially eliminated the response to wounding and petiole girdling. MeJA was almost as effective in N-deficient plants as in those receiving abundant N. Inhibitors of lipoxygenase, the first enzyme in the jasmonic acid biosynthetic pathway, blocked induction by wounding and petiole girdling but not by MeJA. This supports a role for endogenous leaf jasmonic acid (or MeJA) in the regulation of VSP gene expression.     

Inheritance of alleles for Cgy1 and Gy4 storage protein genes in soybean

Summary A cultivar lacking the glycinin subunit A₅A₄B₃ (Raiden) was crossed with one lacking the -subunit of -conglycinin (Keburi). Analysis of F₂ and F₃ progeny indicated that the missing bands of the A₅A₄B₃ and the -subunit were each controlled by a recessive allele of two independently segregating genes. Gene symbols Gy ₄/gy ₄ and Cgy ₁/cgy ₁ were proposed for the genes which confer the presence or absence of the glycinin and conglycinin subunits, respectively.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 9675. Financial support from the American Soybean Association Research Foundation is gratefully acknowledged     

Jasmonic acid-dependent increase in vegetative storage protein in soybean tissue cultures

Adding jasmonic acid (JA) to autotrophic, photomixotrophic, or heterotrophic suspension cultures of soybean specifically increased the level of the M_r 30,000 subunit of soybean vegetative storage protein (VSP-30) and a polypeptide at M_r 18,000 that interacted with antibody raised against VSP. Using photomixotrophic cells, the increase was observed at concentrations as low as 10 nM JA and the increase was evident within 2 h following treatment. Below 10 μM, JA did not inhibit growth of the cells but did cause browning at higher concentrations. Other plant growth regulators, including abscisic acid (ABA), gibberellic acid, and benzyl adenine, did not alter the level of VSP-30 either in the presence or absence of JA. Methyl jasmonate (JA-Me), 3-oxo-2-butyl-cyclopentane-1-acetate, and 3-oxo-2-pentyl-cyclopentane-1-acetate also increased VSP-30 but at higher concentrations than JA. Altering the level of reduced nitrogen or sucrose in the medium did not alter VSP-30 levels in the cells, but at higher sucrose concentrations, sensitivity to JA was reduced. The dramatic increase in VSP-30 elicited by JA appears to be a specific response to the phytohormone.     

Jasmonic acid-dependent increase in vegetative storage protein in soybean tissue cultures

Adding jasmonic acid (JA) to autotrophic, photomixotrophic, or heterotrophic suspension cultures of soybean specifically increased the level of the M_r 30,000 subunit of soybean vegetative storage protein (VSP-30) and a polypeptide at M_r 18,000 that interacted with antibody raised against VSP. Using photomixotrophic cells, the increase was observed at concentrations as low as 10 nM JA and the increase was evident within 2 h following treatment. Below 10 M, JA did not inhibit growth of the cells but did cause browning at higher concentrations. Other plant growth regulators, including abscisic acid (ABA), gibberellic acid, and benzyl adenine, did not alter the level of VSP-30 either in the presence or absence of JA. Methyl jasmonate (JA-Me), 3-oxo-2-butyl-cyclopentane-1-acetate, and 3-oxo-2-pentyl-cyclopentane-1-acetate also increased VSP-30 but at higher concentrations than JA. Altering the level of reduced nitrogen or sucrose in the medium did not alter VSP-30 levels in the cells, but at higher sucrose concentrations, sensitivity to JA was reduced. The dramatic increase in VSP-30 elicited by JA appears to be a specific response to the phytohormone.Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. Paper No. 12474 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.