Isolation and Translation of Plant Messenger RNA 1

A fraction of the RN-A species isolated from Lemna gibba G-3 consists of molecules with attached sequences of polyadenylic acid. This polyadenylic acid-containing fraction, separated from total RNA by adsorption onto oligothymidylic acid-cellulose, was shown to be mRNA by its ability to serve as template in a cell-free translation system derived from wheat germ. The products of translation were characterized by electrophoresis. This method permitted the comparison of mRNA from plants grown under different light conditions. Such plants were shown to possess qualitative and quantitative differences in their mRNA comnplements. Many attempts have been made to discover the level at which specific developmental changes are controlled in plants. Some of these attempts have focused on the possibility of transcriptional control (5, 8, 9, 14-16, 18, 19, 21, 24, 32, 36). Until recently, attempts to isolate and study mRNA from plants depended on the rapid labeling of certain fractions which were not fully characterized (12, 16, 20). With the discovery that most eucaryotic mRNA contains poly(A)3 sequences, a simple procedure for mRNA isolation has become available. Poly(A) RNA has been found now in a number of plant species (10, 13, 23, 30, 33-35) as well as in other eucaryotic organisms (7). We have undertaken to isolate poly(A) RNA from Lemna gibba G-3 and to use a heterologous cell-free translation system to show that this poly(A) RNA is indeed mRNA. In addition, we have tried to develop a general method for examining differences in mRNA populations in plants growing under different conditions. By characterizing the translation products of mRNA one can show differences in the relative content of different messengers as well as in the over-all level of mRNA. Putting duckweed into the dark changes its growth pattern (25, 29). We have chosen initially to compare mRNA from Lemna placed in the dark with the mRNA content after returning the plants to the light. It should now also be possible to make comparisons between the mRNA complement of plants in various developmental stages. 1 Research was supported by National Science Foundation Grant GB-36212. E.M. T. was the recipient of National Institutes of Health Traineeship from Training Grant TI-HD-22 and Institutional Grant RR-07044. 2 Present address: Department of Biology, University of California, Los Angeles, Calif. 90024. 3Abbreviations: poly(A): polyadenylic acid; poly(A) RNA: RNA containing a sequence of polyadenylic acid; oligo (dT) cellulose: oligothymidylic acid covalently linked to cellulose. MATERIALS AND METHODS Lemna gibba G-3 were grown aseptically on E medium (6) in constant light at 22 C. Plants were harvested on Miracloth (Calbiochem), rinsed with distilled H20, and frozen in liquid nitrogen. If the tissue was not used immediately, it was stored at -20 C. Tissue was ground in a Waring blendor in two volumes (v/w) of 0.1 M sodium acetate, pH 5.2, containing 0.2 M LiCI, 1 SDS, and 0.5%O diethylpyrocarbonate (Baycovin, Naftone, Inc., N.Y.) for 2 min at top speed. After adding isoamyl alcohol (1-2%,) to control foaming, the homogenate was extracted with 2 volumes of hot phenol (60 C) containing 1% 8-hydroxyquinoline and saturated with the grinding buffer. The phenol phase was extracted with 0.5 volume of the grinding buffer, and the combined aqueous phases were extracted two more times with phenol. Nucleic acids were precipitated overnight with 2 volumes of cold ethanol, dissolved in 0.01 M tris, pH 7.4, and reprecipitated in the presence of 0.1 M sodium acetate with ethanol. When RNA uncontaminated by DNA was required, the nucleic acid precipitate was dissolved in 0.01 M MgCl2, 0.01 M tris, pH 7.4, and was treated with ribonuclease-free DNase (25 ,ug/rnl [Worthington]) for 1 hr at 37 C. The solution was then brought to 0.01 M EDTA, 0.2 M LiCl, and 0.5% SDS, extracted twice with phenol, and the RNA was precipitated with ethanol. The precipitate was dissolved and reprecipitated in the presence of 4 M potassium acetate. The RNA was further purified in all cases by extraction with 2-methoxyethanol and precipitation with cetyltrimethylammonium bromide, according to the procedure of Bellamy and Ralph (3). Poly(A) containing RNA was isolated on oligo (dT) cellulose columns (2). Total RNA was dissolved in 0.5 M salt (KCI or NaCI) in 0.01 M tris, pH 7.5, and applied to a column containing 0.2 to 0.25 g of oligo (dT) cellulose (T-1, Collaborative Research, Waltham, Mass.). Poly(A) RNA binds to the oligo (dT) cellulose under this high salt condition, but the bulk of the RNA does not bind. The sample was washed onto the column with additional high salt buffer until the eluate did not contain any RNA. The column was then washed with 0.1 M salt, 0.01 M tris, pH 7.5, and the poly(A) RNA was eluted with 0.01 M tris, pH 7.5. The column buffers were made either with KCl or with NaCl and 0.5% SDS. Elution of some poly(A) RNA occurred from some oligo (dT) batches at 0.1 M salt. In some experiments washing with 0.1 M salt was omitted, and the poly(A) RNA was eluted after washing with the original 0.5 M salt buffer. The RNA in fractions from the column was precipitated with ethanol and recovered by centrifugation. In order to demonstrate the existence of poly(A) sequences, RNA was digested with T-1 and pancreatic ribonucleases, which leave poly(A) sequences intact, according to the method of Lee et al (22). After phenol extraction, the poly(A) segment was isolated on oligo (dT) cellulose columns, and its base composition was determined.