The use of alpha-amanitin to inhibit in vivo RNA synthesis and germination in wheat embryos

Abstract

Low concentrations (0.10 to 1.0 microgram/ml) of alpha-amanitin inhibit wheat embryo germination. Labeled RNA, synthesized in vivo by embryos imbibed in the presence of [3H]uridine and various concentrations of alpha-amanitin, was analyzed by oligo(dT)-cellulose chromatograhy and by gel electrophoresis (acrylamide and agarose) coupled with fluorography. Low concentrations of alpha-amanitin (0.10 to 1.0 microgram/ml) strongly and selectively inhibited in vivo poly(A) + RNA synthesis in a manner which closely paralleled alpha-amanitin inhibition of purified RNA polymerase II. These results suggest that de novo mRNA transcription is required for germination. Higher concentrations of alpha-amanitin inhibited in vivo 5 S rRNA and tRNA synthesis in a manner which closely paralleled alpha-amanitin inhibition of purified RNA polymerase III. High concentrations of alpha-amanitin also inhibited accumulation of radioactivity into the rRNA precursor as well as into mature 25 S and 18 S rRNAs in a manner which also closely paralleled alpha-amanitin inhibition of RNA polymerase III. The discrepancy of the in vivo inhibitory effect of high alpha-amanitin concentrations on rRNA synthesis versus a lack of effect on purified RNA polymerase I, which presumably transcribes these genes, can be explained if continued transcription of the large rRNA precursor by RNA polymerase I requires ongoing transcription by RNA polymerase III, or if there is degradation (wastage) of rRNA precursor and/or processed products in the absence of transcription by RNA polymerase III.