Characterization and Complexity of Wheat Developing Endosperm mRNAs

Abstract

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 ...

Gene Expression in Developing Wheat Endosperm

“…The difference was due largely to the accumulation of gliadin mRNAs, which at 14 to 24 DAF may account for 60% of the total poly(A)+ RNA (22). During the early stages ofendosperm development (3-10 DAF), endospermspecific translation products fell into three categories: those that remained relatively constant (enclosed in circles), those that increased (indicated with arrows), and those that decreased (indicated with triangles).…”