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

Abstract

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...

Molecular Characterization of Oat Seed Globulins

“…The partial cDNA clone pOG77, which has the same sequence as the full-length clone pOG2, has previously been used in RNA blot hybridizations to quantitate oat globulin message during seed development (14). In this analysis, globulin mRNA was detectable by 3 Our analysis showed that the amino acid sequence specified by pOG2 is 70% identical to that of the rice globulin (glutelin) reported by Takaiwa et aL (27) (Fig.…”

Oxidative and proteolytic enzyme preparations as promising improvers for oat bread formulations: Rheological, biochemical and microstructural background

“…Oat proteins mainly consist of globulins, which account for up to 75% of total seed proteins (Luthe, 1987). The main storage globulin in oats endosperm is the 12S globulin, a hexameric protein consisting of a (ca.…”

Assessment of the Protein Quality of Two New Canadian-Developed Oat Cultivars by Amino Acid Analysis

“…The primary storage proteins of oats are the globulins, which are found primarily in the cotyledon and axis of the embryo and which account for between 75 and 80% of the total proteins on a dry weight basis (Peterson and Brinegar, 1986; Luthe, 1987; Shotwell and Larkins, 1989; Peterson, 1992). Prolamins, which constitute the other main storage proteins of oats, are found in the endosperm of the seed and account for approximately 10% of the total protein (Shewry and Tatham, 1990).…”