Quiescent Embryos Research Articles

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)(+)RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)(+)RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)(+)RNA content but the initial decrease and subsequent increase in poly(A)(+)RNA levels are less marked. On imbibition at 10°C, the poly(A)(+)RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)(+)RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.

The Isolation of Multiple Forms of DNA-dependent RNA Polymerases from Nuclei of Quiescent Wheat Embryos by Affinity Chromatography

Summary A method is describes which facilitated the rapid mass isolation of nuclei from wheat embryos (wheat germ). Routinely 5 X 107 nuclei/g wheat embryos were obtained. The nuclei showed a DNA: RNA: protein ratio of 1 : 0.3 : 3.5. DNA-dependent RNA polymerases were solubilized from isolated nuclei by sonication in TGED buffer including 0.5 M (NH4)SO4. The isolation of all classes of RNA polymerases was achieved by affinity chromatography on heparin-Sepharose 4B containing 400.ug covalently bound heparin/ml wet gel. Using heparin-Sepharose and DEAE-cellulose chromatography it could be shown that nuclei of quiescent wheat embryos contained all three classes of RNA polymerases (≪the tripartite transcriptive system≫). RNA polymerase II which was further purified showed the typical subunit pattern as revealed by 10–15% acrylamide SDS-gel electrophoresis. The combination of heparin-Sepharose affinity chromatography and conventional chromatography on DEAE-cellulose and DEAE-Sephadex as a general procedure for the simultaneous isolation of all three classes of eukaryotic RNA polymerases is discussed.