Studies on the Nature of Messenger Rna in Germinating Wheat Embryos

Abstract

iDuring the last decade many thorough investigations were carried out on the mechanism of transcription and translation in the living organism. These have clarified the main features of nucleic acid replication and of protein biosynthesis in the cell, and have enabled the study of the early stages of differentiation at a molecular level.'-' Such studies are at present mainly concerned with the structure, function, and changes in the molecular pattern of animal eggs before and after fertilization,4-and of spores and seeds before and after germination.7 Seed germination transfers the embryo from the latent stage into an active developmental stage, and thus represents an interesting phase in the life cycle of the plant. Marcus et al.8 9 have shown that protein synthesis is inactivated both in vivo and in vitro in the ungerminated wheat embryo, which was found to contain ribosomes that could be activated'1 and yielded soluble RNA (sRNA) and supernatant capable of supporting amino acid incorporation. It was thus suggested that messenger RNA (mRNA) is the limiting factor.In the present work, evidence is forwarded for the presence of conserved mRNA in the dry wheat embryo. It is suggested that the masked mRNA is activated upon germination, and supports early protein biosynthesis. Transcription of new mRNA starts at a later stage of development.Materials and Mlethods.-Germination procedure: Wheat embryos (Triticum durumt var. Nursit) were prepared according to Johnston and Stern,1' and stored over CaCl2 at 4°. Viable embryos (500 mg) were germinated in the dark at 230 in a Petri dish containing 5 ml of a germinating medium (GM) containing 0.01 11 tris(hydroxymethyl)aminomethaiie (Tris) buffer, pH 7.6, 0.02 M KCl, sucrose (10 mg/ml), chloramphenicol (50 Ag/ml), and mycostatin (200 ug/ml). Fresh germinating medium was added after 6, 24, 48, and 72 hr germination.Preparation of DNA: DNA was prepared from wheat embryos (3 gm) germinated for 24 hr according to the procedure of Marmur.'2 Preparation of RNA: Wheat embryos were suspended in 0.05 M1 KCl (50 ml) for 2 min at room temperature. Homogenization was performed at 00 in 50 ml T1\I solution (0.01 3l Tris buffer, pH 7.8, 0.01 M1 MgCl2, and 0.023ll KCl), and the RNA was extracted by the phenol method.'3 The RNA obtained was dissolved in TM solution and digested for 4 hr at 280 with deoxyribonuclease (30 ,g/ml Worthington Biochemical Corp., electrophoretically purified). Predigested pronase was then added (200 tg/ml, Calbiochem) and incubation prolonged for 12 hr at 230 in a dialysis bag against 1 liter of TM solution.The final RNA digest was extracted and dissolved in TM solution as above. The DNase and pronase treatments were repeated in the dialysis bag to ensure removal of any DNA and protein contaminants. Final purification of the RNA preparation obtained was effected by methylated albumin kieselguhr (1IAK) column chromatography'4 and filtration through a membrane filter to remove traces of DNA. The purified RNA obtained was 99% sensitive to RNase digestion and insensit...

Changes of Template Activity and Proteins of Chromatin during Wheat Germination

“…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).…”

“…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).…”

Free poly(A) tracts complexed with protein in the cytoplasm of dried wheat embryos

“…It has been shown that the early stages of seed germination are characterized by a rapid increase of the content of polyribosomes in the embryo and by an increase of the rate of protein synthesis [1][2][3][4][5]. The formation of polyribosomes at this developmental stage does not depend on transcription [6].…”

Changes in Ribonuclease Activity of Wheat Plants during Water Stress

“…Important physiological processes altered by water stress include reduced rates of transpiration (Salim and Todd, 1965), translocation (Basler, et al, 1961), respiration (Flowers and Hanson, 1969), photosynthesis (Todd and Basler, 1965), and growth (Chen, et al, 1968).…”