The Major Storage Protein of French Bean Seeds: Characterization in Vivo and Translation in Vitro

The mRNAs for seed lectin and Kuntz tpin inbitor of In addition to substantial biochemical data for these two seed proteins, lectin and trypsin inhibitor have been defined by classical genetic analyses and soybean varieties which lack detectable SBL or SBTI protein have been found (21,22,24). It is the aim of this report and other studies in progress to determine what molecular defects cause the inherited absence of SBL or SBTI protein in certain soybean varieties. Such knowledge will help elucidate the normal functioning and expression of plant genes. As an initial step toward this objective, I have developed modifications to a polysome immunoadsorption procedure which have allowed the isolation of highly enriched mRNAs for lectin and Kunitz trypsin inhibitor. A preliminary report has been published (33). The selected mRNAs were analyzed by their activities in a rabbit reticulocyte cell free translation system and by hybridization of labeled SBL or SBTI cDNA to gel blots of total poly(A) RNA.

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confidence: 98%

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Vodkin¹

1981

“…The cDNA clone pOG77 is representative of only one of several mRNA species present in the developing oat seed (27). At (6)(7)(8)(9)(10)(11)(12) when the 36 and 38 kD polypeptides of the a subunits appeared, and the 22.5 kD polypeptide of the ,-subunit appeared. This upward shift in apparent mol wt was also reported by Robert et al (22,23) and is also shown by immunoprecipitation (Fig.…”

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confidence: 99%

“…Total RNA was isolated from developing seeds using the SDS-proteinase K method of Hall et al (10) except that the RNA was not applied to an oligo dT-cellulose column. Aliquots (4 MIl containing 20 Mg) of RNA were spotted on nitrocellulose that had been pretreated with water and soaked in 20 x SSC (20 x SSC = 3 M NaCl and 0.3 M Na citrate) as described by the manufacturer, (Schleicher and 3Plant Physiol.…”

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confidence: 99%

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Luthe

1987

ABSTRACEOat (Arena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [uSisulfate into protein. Incorporation of r5Sjsulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the a and , globulin subunits were present 2 to 4 DPA, but the full complement ofglobulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.The major storage protein in the oat seed is the salt-soluble globulin, which represents approximately 75% of the total seed protein (6,19). It has many of the same characteristics as the 11 to 12 S globulins of other seeds, including legumes (1,2,3,26). Recent DNA sequence analysis indicated that the , subunit of oat globulin was about 40% homologous with that of legumes (27). Of all the cereals studied, only oat and rice have these 'globulin-like' proteins as their major storage protein fraction (19,29 (17) medium without added phytohormones (0-MS). After the label was taken up, it was chased for 3 h with 0-MS. Seeds were then removed from the panicle, dehulled, and lyophilized.Extraction of Seed Proteins. Total seed protein was extracted by homogenizing 10 seeds from each sampling date in 2 ml of sample buffer (11) containing 1 mm phenylmethyl sulfonyl fluoride, boiled for 5 min and centrifuged at 13,000g (13). Globulin was extracted by homogenizing five seeds in 2 ml of globulin extraction buffer (6, 18). To determine the amount of 35S incorporated into total protein and globulin, aliquots of each extract were spotted on Whatman 3MM discs, TCA-washed and counted as described previously (13). Results reported are the average of duplicate samples.Immunoprecipitation. Aliquots (0.5 ml) of the globulin extract were mixed with 3 ml of PBS (10 mm Na phosphate buffer (pH 7.2) in 0.145 M NaCl), 317 ,ug of anti-oat globulin IgG (immunoglobulin G) purified on DEAE-Affigel Blue (BIORAD) (25) was added, and the mixture was incubated for 2 h at 37C followed by incubation at 4°C overnight. The next morning the tubes were warmed to 37°C and IgG-SORB (The Enzyme Center Inc., Boston, MA) was added according to the manufacturer's recommendations and incubated for 2 h at room temperature. Following the incubation the samples were washed with NET-2 (12) four times and with water one time. Samples were dissolved in...

“…We also show that the amino acid sequence predicted by the oat globulin cDNA is more similar to that of the globulin (glutelin) of rice seeds than to the 1 S globulins of legumes. (10). The RNA was suspended in 10 mM Tris-HCl (pH 7.5) and 500 mm KCI, and poly(A)-containing RNA was obtained by one cycle of oligo(dT)-cellulose chromatography (3).…”

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confidence: 99%

Molecular Characterization of Oat Seed Globulins

Shotwell

Afonso²,

Davies³

et al. 1988

We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a higly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).During their development, plant seeds accumulate large amounts of storage proteins that serve as sources of nitrogen, sulfur, and carbon compounds during seed germination (25). Two major classes of storage proteins can be distinguished: globulins, which are found principally in the cotyledons and axis of the embryo, and prolamines, which are found in the endosperm of cereal seeds (13). Two major types of storage globulins have been described that have sedimentation coefficients ofabout 7S and 11S. The proportion of the two types of globulins is variable among dicots; monocot embryos contain only the 7S globulin, which is present in the scutellum.Storage globulins account for most of the protein in dicot seeds, but they generally make up only a small fraction of the protein found in cereal seeds. Instead, most cereals contain predominantly prolamine-type storage proteins. Oats and rice are exceptions. These two contain only small amounts ofprolamine (5-10%), and most of their storage protein is an 11 to 12S globulin that is synthesized in the endosperm. Both of these proteins are structurally related to the 11S globulins found in dicots (25), but both are much less soluble than the dicot 11S globulins. The oat globulin requires 0.8 to 1.0 M NaCl for solubility, whereas the characteristics of the rice globulin (glutelin) are such that denaturing solvents are required for solvation.Previous studies in our laboratory (29) and elsewhere (5, 7)