Exogenous methionine depresses level of mRNA for a soybean storage protein

$\text{In vitro}$ translation experiments indicate that absence of the β-subunit of 7S storage protein in soybean ($\text{Glycine max}$ L. Merr. cv. Provar) cotyledons cultured on methionine-supplemented medium is due to lack of functional mRNA for that polypeptide. Relative amounts of functional mRNA for the 7S α′- and α-subunits were unaffected by methionine in the cotyledon culture medium. Measurements of β-subunit accumulation in cotyledons transferred from basal medium to methionine-supplemented medium show that methionine inhibits continued accumulation of the β-subunit after synthesis of the β-subunit has begun, and that methionine does not promote degradation of existing β-subunit.     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

Storage Protein Composition of Soybean Cotyledons Grown In Vitro in Media of Various Sulfate Concentrations in the Presence and Absence of Exogenous l-Methionine

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

Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia

The 7S seed storage protein (β-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; α, α′, and β. Accumulation of the β-subunit, but not the α- and α′-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the α′- or β-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the β-subunit gene is repressed by exogenous methionine in in vitro cultured seeds, whereas the α′-subunit gene expression is not affected; and (b) accumulation of the β-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major seed storage protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major seed storage proteins.     

beta-Conglycinins in Developing Soybean Seeds

The temporal sequence of development of the major proteins of seeds of soybean (Merr.) has been studied during development of cotyledons from flowering to maturity. A well-defined difference occurred in the times of appearance and the periods of maximum accumulation of α, α′-, and β-subunits of betaconglycinin. Whereas α- and α′-subunits appeared 15 to 17 days after flowering, accumulation of β-subunit did not commence until 22 days after flowering. Such alterations in subunit composition infer that changes also occurred in the amino acid composition of betaconglycinin during maturation, particularly in the content of methionine which is low in the β-subunit.     

The development of isocitric lyase activity in germinating cotton seed

In cotyledons of germinating cotton (Gossypium hirsutum L. var. Stoneville 213) seedlings, in the dark, isocitric lyase (EC 4.1.3.1) activity peaks after 2 days and thereafter slowly declines to a negligible value after 8 days. The maximum activity of this enzyme in cotyledons of 2-day-old seedlings was 16.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, completely prevents the development of isocitric lyase activity in these germinating seed. In cotyledons of germinating cotton seedlings, in the light, isocitric lyase activity peaks after 2 to 3 days and sharply declines to a negligible value after 4 days. The maximum activity of this enzyme in cotyledons of 2- to 3-day-old seedlings was 13.2 μmoles of glyoxylate formed/15 min·10 cotyledon pairs. Actinomycin D at a concentration of 10 μg/ml, if added to the imbibing solution, severely inhibits the development of enzyme activity.     

Biosynthesis of alpha-Amylase in Vigna mungo Cotyledon

In vitro translation of RNA extracted from Vigna mungo cotyledons showed that α-amylase is synthesized as a polypeptide with a molecular mass of 45,000, while cotyledons contain a form of α-amylase with a molecular mass of 43,000. To find out whether the 45,000 molecular mass polypeptide is a precursor to the 43,000 found in vivo, the cell free translation systems were supplemented with canine microsomal membrane; when mRNA was translated in the wheat germ system supplemented with canine microsomes, the 45,000 molecular mass form was not processed to a smaller form but the precursor form was partly processed in the membrane-supplemented reticulocyte lysate system. When V. mungo RNA was translated in Xenopus oocyte system, only the smaller form (molecular mass 43,000) was detected. Involvement of contranslational glycosylation in the maturating process of the α-amylase was ruled out because there was no effect of tunicamycin, and the polypeptide was resistant to endo-β-H or endo-β-D digestion. We interpret these results to mean that the 45,000 molecular mass form is a precursor with a signal peptide or transit sequence, and that the 43,000 molecular mass is the mature form of the protein.     

Free Amino Acid and Storage Protein Composition of Soybean Fruit Explants and Isolated Cotyledons Cultured with and without Methionine

Thein vitroculture of immature soybean cotyledons (in direct contact with the medium) and immature fruit explants (stem dipping into the medium) on a defined medium containing glutamine and sulphate as sole sources of N and S for 7 d led to rates of growth and reserve protein accumulation close to, or greater than, those occurringin situ. Supplementation of the medium with 8.4 mMmethionine had little effect on growth and protein accumulation of the cotyledons in the explant system, but did result in significant increases in the isolated cotyledon system. Methionine suppressed the synthesis of the 7S β-subunit in both systems. The free amino pool of the cotyledons increased more than three-fold when methionine was present in the explant medium. In the isolated cotyledon system, the basal medium alone caused a large increase (over 30-fold) in the free amino acid fraction, but methionine resulted in an even greater increase (over 50-fold). In both systems the expansion involved a very large increase in the methionine pool, but many other amino acids also showed large increases. Specific effects of methionine on individual amino acids were more clear in the explant system, where its presence resulted in marked increases in serine, alanine and asparagine. The data show that an abnormal situation arises on feeding with methionine, a fact to be considered before attributing effects on growth and protein synthesis directly to methionine.     

Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qβ replicase, together with EF-Tu and EF-Ts, for Qβ RNA replication in E. coli. We analyzed the crystal structure of Qβ replicase, consisting of the virus-encoded RNA-dependent RNA polymerase (β-subunit), EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qβ RNA replication. Structural and biochemical studies revealed that the two N-terminal OB-folds of S1 anchor S1 onto the β-subunit, and the third OB-fold is mobile and protrudes beyond the surface of the β-subunit. The third OB-fold mainly interacts with a specific RNA fragment derived from the internal region of Qβ RNA, and its RNA-binding ability is required for replication initiation of Qβ RNA. Thus, the third mobile OB-fold of S1, which is spatially anchored near the surface of the β-subunit, primarily recruits the Qβ RNA toward the β-subunit, leading to the specific and efficient replication initiation of Qβ RNA, and S1 functions as a replication initiation factor, beyond its established function in protein synthesis.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.     

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB_1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB₂ domain, and use NMR spectroscopy to demonstrate for the first time that OB₂ is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.     

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the α- and β- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t_½ = 50 versus 150 minutes, respectively) and during the deinduction period (t_½ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd.]

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45 μM 2, 4-D, 2.32 μM Kin and 15 mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15 mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22 μM BAP.     

Effects of nutritional stress on the storage proteins of soybeans

The effects of sulfur deficiency on the complement of proteins laid down in developing seeds of soybean (Glycine max L. Merr) have been examined. Sulfur deficiency caused a 40% decrease in the level of glycinins and a contrasting elevation in the level of β-conglycinins. The subunit composition of these proteins was also affected. There was in particular a 3-fold increase in the β-subunit of β-conglycinins in the sulfur-deficient seeds, and this accumulated largely as the B₀-isomer of β-conglycinins, a protein which while virtually devoid of methionine and cysteine retains the physical properties of a normal 7S storage protein. These data demonstrate that a high degree of selectivity can be exerted by environmental stress over the accumulation of proteins in developing seeds.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

In Vitro and In Vivo Oxidation of Methionine Residues in Small, Acid-Soluble Spore Proteins from Bacillus Species

Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H₂O₂). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H₂O₂ caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H₂O₂ than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.     

Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (α-subunit) and 65 (β-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in β-subunit and twofold increase in α-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total β-subunit to α-subunit content. The β- to α-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.     

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control

Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different.