Sugar uptake by cotton tissues: leaf disc versus cultured roots

The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.

Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The α and β globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The α and β subunits appeared during the same chase period and assembled into a 12S oligomer.

The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the α and β subunits forming a 12S oligomer.

     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

Synthesis of Oat Globulin Precursors : Analogy to Legume 11S Storage Protein Synthesisa

The oat (Avena sativa L.) seed globulin was found to be synthesized in vitro as 60,000 to 64,000 dalton precursors. In vivo protein labeling yielded polypeptides of 58,000 to 62,000 daltons, suggesting cleavage of signal sequences from the precursors. Further cleavage is apparently required to separate the α and β polypeptide sequences which are known to form disulfide-linked 53,000 to 58,000 dalton species in the (αβ)₆ holoprotein. The data are discussed with respect to analogous synthesis and processing of some legume 11S storage proteins.     

Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.     

A Seed Storage Protein with Possible Self-Affinity through Lectin-Like Binding

The primary storage protein of oat (Avena sativa L.) seeds, globulin, was shown to have a specific carbohydrate-binding activity. The globulin was capable of hemagglutinating rabbit red blood cells and this hemagglutination was inhibited by the β-glucan, laminarin, as well as by carbohydrate which had been cleaved from the native globulin. Globulin with carbohydrate-binding activity was isolated from cell wall preparations and from defatted flour. The lectin activity apparently resides in the α-subunit of the globulin and has affinity for the carbohydrate which is O-glycosidically linked to the globulin. A portion of this carbohydrate is attached to the β-subunit. Two affinity columns were synthesized utilizing laminarin and the carbohydrate from the native globulin as ligands. The hemagglutinating activity bound to both of these columns. The activity was specifically eluted from the globulin-carbohydrate affinity column with carbohydrate cleaved from native globulin by an alkali-catalyzed β-elimination. The possible roles of this unique self-binding capacity are discussed.     

Separation and characterization of oat globulin polypeptides

The storage globulin of oat seeds was separated into its acidic (α) and basic (β) polypeptides by ion-exchange chromatography in 6 m urea and further characterized by several electrophoretic techniques. Molecular weights of the α and β polypeptides were 32,500–37,500 and 22,000–24,000, respectively. The unreduced protein existed as disulfide-linked αβ species of molecular weight 53,000–58,000. Isoelectric points were approximately 5.9–7.2 (α) and 8.7–9.2 (β). Two-dimensional electrophoresis showed considerable heterogeneity within both groups of polypeptides. More complete amino acid analyses of the globulin and its polypeptides are presented along with a proposed structure of the native protein based on previous and present data. Similarities were noted between the oat globulin and the legumin (11 S) class of storage proteins in certain legumes.     

Protein Synthesis and Accumulation in Bean Cotyledons during Growth

Analysis of total protein, of specific proteins by gel electrophoresis and immunoelectrophoresis, and of protein synthetic activity in vitro confirmed that intense protein synthesis and accumulation occurred as the French bean (Phaseolus vulgaris L). seed grew from 12 to 20 millimeters. These techniques showed that there was no globulin-1 (G1) fraction (requiring high salt for solubility) present in 6-millimeter seeds, and only very small amounts were synthesized in seeds less than 9 millimeters long. The 7- to 9-millimeter stages represent a 2-day transition period over which genetic information for the G1 protein becomes actively expressed, accounting for at least 50% of all protein synthesized in this tissue during the following 14 days. At maturity, the electrophoretic analysis confirmed that G1 globulin was the major storage protein, representing some 50% of the dry seed protein. Cell-free protein synthesis assays, including immunoprecipitation of the in vitro products, clearly showed G1 polypeptides to be among the polysome-directed products.     

Abscisic Acid localization and metabolism in barley aleurone layers

Aleurone layers of Hordeum vulgare, cv. `Himalaya' took up [¹⁴C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4′-dihydrophaseic acid (DPA) and three polar metabolites Mx₁, Mx₂, and Mx₃. ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue-specific. Layers metabolized [³H]DPA to Mx₁ and Mx₂. ABA, PA, and DPA-methyl ester and epi-DPA-methyl ester inhibited synthesis of α-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds.     

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 μm, was labeled to radioisotopic equilibrium with ³⁵SO₄²⁻. Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. γ-Glutamylcyst(e)ine, erythro- and threo-thiothreonine, and S-methylcysteine were not detected. No volatile ³⁵S compounds were formed during plant growth at 1,000 μm sulfate, nor were significant amounts of ³⁵S compounds excreted into the medium.     

Quantitation of oat globulin by radioimmunoassay

A radioimmunoassay was used to determine the globulin content of developing oat (Avena sativa L. var Coker 6622) seeds. The globulin content increased from 0.32 to 2.37 milligrams per seed from 2 to 21 days post anthesis. The amount of globulin did not increase from 21 to 30 days post anthesis. When the total protein of seeds harvested 21, 24, and 30 days post anthesis was measured by micro-Kjeldahl analysis, it was determined to be 3.15 milligrams per seed for each sampling date. When the amount of globulin per seed was compared to this value, the relative proportion of globulin to total seed protein was approximately 75%.     

Structural Relationship among the Rice Glutelin Polypeptides

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the α and β subunits are proteolytic products of the 51 kD precursor protein. The poly(A)⁺ RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.     

Analysis of globulin maturation in developing sunflower seeds

The synthesis of major storage globulin polypeptides has been examined in developing seeds of sunflower(Helianthus annuus L.). Analyses of total proteins and purified globulins, also called helianthinin, by gel electrophoresis and immunoelectrophoresis have shown that a burst of protein synthesis and accumulation occurs around 10 d after flowering. There is no mature globulin before that time and only small amounts of precursor forms can be detected. Thus, 10–12 d after flowering appears to be a transition period during which genetic information for the globulin becomes actively expressed. Immunoelectrophoresis has confirmed that globulin is the main storage protein, at seed maturation, accounting up to 70 % of total proteins per kernel. Pulse chase experiments have shown that synthesis initially involves the formation of high molecular mass precursors and that storage proteins are post-translationally processed. Intermediary products, with molecular mass higher than early translational products, can be detected, together with mature globulin polypeptides.     

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

Influence of Sulfur Nutrition on Developmental Patterns of Some Major Pea Seed Proteins and Their mRNAs

In addition to the marked reduction in legumin synthesis and legumin mRNA levels reported earlier (Chandler, Higgins, Randall, Spencer 1983 Plant Physiol 71: 47-54), pulse labeling of S-deficient Pisum sativum L. seeds showed that a high relative level of total vicilin (vicilin plus convicilin) synthesis was maintained throughout the entire phase of protein accumulation, whereas in nondeficient seeds vicilin synthesis is largely confined to the first half of this phase. Fractionation of pulse-labeled proteins on Na-dodecylsulfate-polyacrylamide gels showed that the synthesis of the M_r 50,000 family of vicilin polypeptides was increased and greatly extended in S-deficient seeds whereas that of convicilin was slightly reduced. Other changes apparent from pulse-labeling experiments include a depression, to different degrees, in the synthesis of three major albumin polypeptides.

The level of the mRNAs for seven major seed proteins was followed throughout development of control and sulfur-deficient seeds. In all cases, the changes in each mRNA closely reflected the pattern of synthesis of its corresponding polypeptide seen by pulse labeling. S-deficient seeds showed an elevated level of M_r 50,000 vicilin mRNA which remained high throughout seed formation, whereas legumin mRNA levels were greatly reduced at all stages of development.

When S-deficient plants were given an adequate supply of sulfate midway through seed development, there was a shift toward the protein synthesis profile characteristic of healthy plants. The synthesis of legumin and two albumins rapidly increased and the synthesis of M_r 50,000 vicilin declined more slowly. Similar responses were seen in detached, S-deficient seeds supplied directly with adequate sulfate.