Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Abstract

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5-and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.Protein synthesis has been clearly established as an obligatory early component of seed germination (7,19,32,33 MATERIALS AND METHODSPreparation and Germination of Embryos. Embryos were prepared from wheat seed (Triticwn aestivwn L.) by the procedure of Johnston and Stern (13) using either CCl4-Cyclohexane flotation (20) or sucrose flotation to separate the embryos from the endosperm. The embryos obtained by the sucrose flotation procedure are referred to as "sucrose" embryos. Germination was carried out as previously described (23) Ribosome Isolation. Two 125-mg portions of embryos frozen in dry ice were converted separately into "dry ice powders" (22) and then combined for homogenization in 7 ml of isolation media (0.25 M sucrose, 20 mm KCI, 100 mm tris-HCl [pH 7.6], 5 mM magnesium acetate, 5 mM mercaptoethanol) in a Duall conical glass homogenizer. The homogenate was cleared by centrifugation (23,000g for 10 min) and layered over 2 ml of a cushion of 1.8 M sucrose, 20 mm KCI, 5 mM magnesium acetate, 100 mM tris-HCl (pH 7.6), 5 mm mercaptoethanol, and centrifuged for 90 min at 150,000g in a Spinco Ti5O rotor. The resulting pellet was resuspended in 0.6 ml of 20 mm KCI, 5 mM magnesium acetate, 50 mm tris-HCl (pH 7.6), 1 mm dithiothreitol and clarified by centrifugation for 10 min at 23,000g. The ribosome content was determined by measuring the absorbance at 260 nm of an aliquot (10 A26o = 1 mg of ribosomes). For fractionation, a volume containing 340 ,g of ribosomes was layered on 4.5 ml of a 15 to 38% linear sucrose gradients over 0.5 ml of a 1.5 M sucrose cushion (both gradient and cushion solutions contained 20 mi KCI, 5 mm magnesium acetate, 50 mm tris-HCI [pH 7.6], 1 mM dithiothreitol) and centrifuged for 45 min at 150,00Og in a SW50.1 rotor. In the EDTA release experiment, a volume containing 540 ,ug of ribosomes was layered on 5 ml of a 5 to 21% linear sucrose gradient containing 50 mm KCI, 50 mm tris-HCI (pH 7.6), 10 mm potassium EDTA (pH 7.4), and centrifuged for 80 min at 150,000g in a SW50.1 rotor. The gradients were fractionated with continual monitoring of absorbance at 254 nm; the fractions were ...

Changes of Template Activity and Proteins of Chromatin during Wheat Germination

“…in wheat embryos (6). Spiegel et al (18) hr; 11.9 ,ug, 24 hr; 10.7 ,ug. 36 hr; 9.9 ,Lg, 48 hr; 10.5 ,.cg, 60 hr.…”

“…Investigation of the incorporation of precursors for RNA into acid-insoluble materials in wheat embryos showed that syntheses of mRNA, rRNA, and tRNA were activated and increased as a function of germination periods (6,7,18). Particularly, an experiment using :'2Pi as a precursor indicated that RNA synthesis in wheat embryos up to 24 hr of germination was very low, compared with that between 24 and 48 hr (6).…”

“…It was essentially constant for 18 hours of germination, corresponding to 2.5% of that of a native calf thymus DNA. Thereafter, the activity increased 2-fold and 5-fold at 24 and 60 hours of germination, respectively.Chromatin Investigation of the incorporation of precursors for RNA into acid-insoluble materials in wheat embryos showed that syntheses of mRNA, rRNA, and tRNA were activated and increased as a function of germination periods (6,7,18). Particularly, an experiment using :'2Pi as a precursor indicated that RNA synthesis in wheat embryos up to 24 hr of germination was very low, compared with that between 24 and 48 hr (6).…”

Newly Synthesized mRNA Is Translated during the Initial Imbibition Phase of Germinating Maize Embryo

“…The results suggest that translation of newly synthesized mRNA occurs after a delay of several hours (4,5,7,11 molecules (27). In this material, the initial water uptake is completed after 30 min (19).…”

Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos

“…Differences In wheat embryos the activities to synthesize mRNA, rRNA, and tRNA were observed to be activated and increased during germination (8,9,11,25,29, 34 (35) reported that the template activity of chromatin with exogenous DNA-dependent RNA polymerase II from wheat embryo was constantly very low for the first 18 h of germination, thereafter increased 2-fold and 5-fold at 24 h and 60 h of germination, respectively, in parallel with the decrease in the amounts of two major nonhistone proteins, mol wt 39k and 59k polypeptides. We have recently demonstrated that some nonhistone proteins, different from 39k and 59k polypeptides, which originated from germ chromatin having very low template activity, inhibited transcriptional activity; whereas those which originated from seedlings having high template activity stimulated transcriptional activity in reconstituted chromatin (36).…”