In vitro synthesis of barley storage proteins

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA' RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)' RNAs, analyzed on isokinetic sucrose gradient with [Hjpolyuridylic acid [poly(U)i hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin) 2Abbreviations: MB, membrane-bound; poly(A), polyadenylic acid; poly(U), polyuridylic acid; MB poly(A)+ RNA, polyadenylated RNA extracted from membrane-bound polysomes. plant gene expression: they are specifically synthesized in the endosperm, and nowhere else; all the different gliadins are synchronously synthesized (19), whereas the corresponding genes are located on different chromosomes (2, 10, 11, 21). Moreover, the endosperm of developing wheat seed is an example of a differentiated eukaryotic cell system in which a relatively small number of proteins, the seed storage proteins, are synthesized to a very much greater amount than other proteins. In other similar developmental cell systems, mRNA species could be divided into a series of abundance classes by a study of the kinetics of their hybridizations to homologous cDNAs (6).The present paper describes experiments intended to evaluate the extent of genetic information expressed in the developing wheat endosperm, i.e. to investigate the complexity of the mRNAs extracted either from free or from MB polysomes, isolated when the in vivo gliadin synthesis rate is maximal (19). MATERIALS AND METHODSReagents. Radiochemicals were purchased from Amersham and New England Nuclear. Reverse transcriptase was obtained from Dr. J. W. Beard, (Life Science, St. Petersburg, FL), RNasin from Genofit (Geneva, Switzerland), and the wheat germ in vitro translation system from BRL. All other reagents were from Sigma Chemicals Co. and Merck. Reagents were RNAse free grade. Otherwise specified, all procedures were carried out at 4°C using heat-sterilized glassware. The distilled H20 utilized for the preparation of solutions was boiled in the presence of 0.05% (v/v) diethylpyrocarbonate.Plant Materials. Developing wheat seeds (Triticum aestivum cv Cappelle-Desprez) were harvested 25 d after anthesis directly into liquid nitrogen and stored at -80°C until required.Preparation and Analysis of Polysomes. Frozen endosperms (fresh weight, about 25 g for 400), dissected free from embryos, were ground in liquid nitrogen and mixed to 100 ml of cold extraction buffer containing 200 mM Tris-HCI (pH 9), 100 mm KCI, 35 mM Mg-acetate, 5 mm 2-mercaptoethanol, 25 mM EGTA, 200 mm sucrose. After an 8-min centrifugation at 700g, the supematant was centrifuged again for 10 min at 30,000g in a Sorvall SS 34 rotor. The pellets were dissolved in 10 ml of extraction buffer containing 0.5% (w/v) Triton X-100 and recentrifuged for 10 min at 30,000g. Free polysomes (first supematant) and initially MB polysomes (second supernatant) were ...

Section: Discussionsupporting

confidence: 83%

Characterization and Complexity of Wheat Developing Endosperm mRNAs

Pernollet¹,

Vaillant²

1984

“…The seed proteins of the grain, synthesized during seed development in the endosperm and/or aleurone layer, include the hordeins (30-50% of total N, [17]), and a diverse group of salt-soluble proteins (20-40% total N, [9]). The hordeins, which function as storage polypeptides, are synthesized on RER and accumulate in protein bodies in starchy endosperm cells (17). The salt-soluble proteins have more active physiological functions.…”

mentioning

confidence: 99%

Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

Mundy¹,

Hejgaard²,

Hansen³

et al. 1986

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA3. B and C hordein polypeptides and the salt-soluble proteins O-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-I and 2), the a-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, f6-amylase, protein Z, and CI-1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-I and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-I survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-I, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of a-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for a-amylase, these hormones have the opposite effect on ASI mRNA levels.One approach to understanding the molecular mechanisms controlling seed maturation and germination is to study organspecific, developmentally characteristic, and hormonally regulated seed proteins and their mRNAs. Earlier studies have identified such polypeptides in Gossypium (7) and Brassica (6) and have formed the basis of later work at the molecular level on these crops. We report here the results of similar experiments with barley seeds, a cereal of importance as livestock feed and as malt in the brewing industry.The barley seed or caryopsis is comprised of several tissues derived from the fertilized ovule (4). The seed proteins of the grain, synthesized during seed development in the endosperm and/or aleurone layer, include the hordeins (30-50% of total N, ' Present address:

“…Several storage protein polypeptides of cereals (4,8,16,20) and legumes (9,11,24,28) are synthesized as short-lived precursor polypeptides which may undergo modification during or after translation. Some of these precursors seem to be the equivalents of precursor polypeptides of secretory or membrane proteins in that a 'signal' peptide may be present on the polypeptide (2).…”

mentioning

confidence: 99%

Biosynthesis of Storage Proteins in Developing Rice Seeds

Yamagata

Sugimoto²,

Tanaka³

et al. 1982

289

203

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10-to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76-and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with I14CIleucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kllodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.The major storage proteins of most cereal grains are glutelin and prolamin. In rice grains, however, glutelin is the major protein of the starchy endosperm, constituting at least 80%o of the total protein, prolamin accounting for less than 5% (12). Rice glutelin has a mol wt of 6 x I05 according to Tecson et al. (27), but Takeda et a!. (25) demonstrated that it has a heterogeneous mol wt. The increase in glutelin in the developing rice grain coincides with the appearance of PB' in the starchy endosperm 7 to 8 DAF (7,29), and glutelin is found exclusively in PB (7,26,30). In a previous paper (26), we reported two types of PB in the starchy endosperm of rice grains and described their isolation. One (type I, PB-I) is spherical with a concentric ring structure, whereas the other (type II, PB-II) is stained homogeneously by osmium tetroxide and does not have this structure. PB-I contains prolamin, and PB-II is rich in glutelin and globulin. The glutelin in PB-II is composed of two principal subunits, the 22-to 23-and 37-to 39-kD complexes, and the prolamin in PB-I is composed mainly of 13-kD polypeptide.