The Turnover of Nucleic Acids in Lemna minor

Abstract

A method is described for measuring the rate constants of both synthesis and degradation of nucleic acids in sterile growing cultures of Lemna minor which avoids the difficulties of environmental changes in isotope uptake and precursor pool size. In fast growing cultures the half-life of riboso-mal RNA has been estimated to be between 5 and 8 days. This half-life has been shown to consist of two components , cytoplasmic ribosomal RNA with a half-life of about 4 days and chloroplast ribosomal RNA with a half-life of about 15 days. The possible interference of recycling has been checked, and the evidence indicates its likely insignificance. "Heavy" labeling of Lemna with D20 and 16NO-g has provided evidence for the conservation of ribosomal RNA in fast growing cultures and has also provided an alternative assessment of recycling. When Lemna is placed on water, the rate of degradation of ribosomal RNA is increased and that of synthesis is decreased. Under partial "step down" conditions it has been found that omission of either nitrate or phosphate, or calcium or magnesium leads to an increase in the rate of degradation of ribosomal RNA. In Lemna grown on water, benzyladenine increases both the synthetic and degradative rates of nucleic acid metabolism. Abscisic acid, on the other hand, markedly reduces the rate of synthesis of ribosomal RNA but leaves the degradative rate unaltered. conditions on the following medium: sucrose, 10 mM; Ca (NO3)2-4H20, 5 mm; KNO3, 5 mm; KH2PO4, 2 mM; MgSO4M7H20, 2 mM; H3BO3, 0.05 mM; MnCl2 4H20, 0.01 mM; CuSO4-5H20, 0.03 ;iM; FeSO4, 0.05 mM; EDTA Na2, 0.05 mM; and pH adusted to 5.0. Cultures were grown under a constant light intensity of 103 ft-c (F 10%) obtained from mixtures of warm white and daylight fluorescent light tubes at 25 C. Cultures were checked for microbial contamination at each transfer by placing several fronds from each culture on 2% sucrose-nutrient broth (Oxoid, Ltd.) and incubating at 30 C for 7 days. Absence of cloudiness was accepted as evidence for sterility. In cases of uncertainty, portions of the nutrient broth were streaked on 1% nutrient agar and, after further incubation, examined for colonies. All nonsterile experiments were discarded. CsCI and Cs2SO4. Reagent grade chemicals were obtained, boiled with acid-washed charcoal, and recrystallized from hot water after filtration. The A42 of a saturated solution of CsCl was 0.028 and of CS2SO4 0.06. Radiocbemicals. 8H-2,8-Adenine (3 c/mmole) and 14C-8-adenine (35 mc/mmole) were obtained from the Radiochemical Centre, Amersham. Before use the 'H-adenine was purified by one-dimensional chromatography on Whatman No. 4 paper with isopropanol-H20-concentrated hydrochloric acid (130:37: 33). In the same system the "C-adenine had a purity of 99.7% and was therefore used without chromatographic purification. H15NO3, 96.5% was obtained from Office National Industrial de I'Azote, France. Ca (15NOS)2 and K15NO3 were prepared from aliquots of the nitric acid by neutralization with CaC03 or KOH. Abscisic acid was the kind gift of Dr. R. W. A. Leach of the Woodstock Agricultural Research Centre, Sittingbourne, Kent. Turnover in nucleic acids in animal systems is a well established phenomenon and has been so for many years (8). In contrast, evidence concerning the stability of nucleic acids in plant systems is infrequent. Ingle and Key (3) and Loening (4) have demonstrated rapid turnover of certain nucleic acid fractions of probable nuclear origin. In other cases, excised plant tissues frequently exhibit losses of RNA (13). These data cannot be construed, however, as evidence for degradation of nucleic acids in growing plants since bacteria exhibit nucleic acid stability under conditions of normal growth but readily exhibit degradation when grown under step down conditions (8). The stability of the major species of nucleic acids in growing plants is therefore largely unknown. The work described in this paper was instituted to determine the stability of the major species of nucleic acids, in particular ribo-somal RNA, in plant tissues. Some of the factors which control either degradative or synthetic rates of nucleic acids in plants are considered in this paper. MATERIALS Plant Material. Single fronds of Lemna minor were obtained from local sources and sterilized by treatments of about 30 sec with 6% sodium hypochlorite. L. minor was grown under sterile METHODS Isolation and Separation of Nucleic Acids. The fresh tissue was ground in a pestle and mortar with equal volumes of 1% sodium lauryl sulfate and 80% phenol-10%0 m-cresol-0.01 % 8-hydroxy-quinoline at room temperature (6). After stirring for 10 min the aqueous phase was made 3% with respect to NaCl and stirring continued for another 5 min. After centrifugation, the upper phase was removed and precipitated with 2 volumes of ethanol. The precipitate was washed with 80% ethanol, dissolved in freshly prepared tris, 0.08 M; sodium acetate, 0.04 M; sodium EDTA, 4 mm, pH 7.8, containing 0.2% sodium lauryl sulfate, and reprecipitated with ethanol. Subsequent purification was carried out by dissolving in tris-acetate-EDTA and adding equal volumes of 2.5 M potassium phosphate (pH 8.0) and methoxyethanol (9). After mixing and centrifugation, the upper phase was removed and mixed with 3 volumes of tris-acetate-EDTA containing 0.1% cetyltrimethyl-ammonium bromide. After standing at 0 C for 15 min, the precipitate was spun down and washed twice with 70% ethanol containing 0.1 M potassium acetate (pH 5.0) (9). The product obtained at this stage was of high spectral purity with an A2O/23 of 2.35 to 2.45 and an A2J2w of 2.2 to 2.24. The 41° was 230. 742