In-vitro translation of messenger-RNA from developing bean leaves. Evidence for the existence of stored messenger-RNA and its light-induced mobilisation into polyribosomes

Abstract

Poly(A)-containing messenger RNA was purified from polyribosomes isolated from the primary leaves of 7-day-old dark-grown seedlings of Phaseolus vulgaris var. Masterpiece. Analysis of the messenger RNA on 2.4% polyacrylamide gels showed that it consists of a heterogeneous population of molecules with an average molecular weight of 500,000. The nucleotide composition of the RNA was 16.0% cytidylic acid, 39.4% adenylic acid, 21.3% guanylic acid and 23.2% uridylic acid. Based on the degree of resistance of the RNA to digestion with ribonucleases A and T1 the average length of the poly(A) sequence was calculated to be 120 nucleotides. No significant differences in mobility in polyacrylamide gels, nucleotide composition or polyadenylic acid content were found between the poly(A)-containing mRNA from polyribosomes of primary leaves of dark-grown plants and those given a 16 h white light treatment. Purified poly(A)-containing mRNA was shown to direct the incorporation of [(35)S]methionine into proteins in an in vitro protein-synthesising system from wheat germ. The protein products were fractionated according to molecular size by electrophoresis in 15% polyacrylamide/urea/SDS gels and the protein bands were detected by fluorography. Messenger RNAs directing the synthesis of three polypeptides with molecular weights of 34,000, 32,000 and 25,000 were detected in polyribosomes of plants following white light treatment. These messenger RNAs were absent, or present in much lower amounts, in polyribosomal messenger RNA from leaves of dark-grown plants, although they were present in total cell poly(A)-containing RNA. This indicates that certain messenger RNAs may be stored in the dark and that light stimulates these RNAs to engage in polyribosome formation. Continuous far-red (730 nm) irradiation for 4 h also caused the appearance of these messenger RNAs in the polyribosomes although 5 min red light followed by 4 h darkness had little effect. This suggests that phytochrome acting in the "high energy" mode, may be the photoreceptor responsible for initiating the response.