Heterotrophic n(2) fixation and distribution of newly fixed nitrogen in a rice-flooded soil system.

Rice (Oryza sativa L.) plants growing in pots of flooded soil were exposed to a (15)N(2)-enriched atmosphere for 3 to 13 days in a gas-tight chamber. The floodwater and soil surface were shaded with a black cloth to reduce the activity of phototrophic N(2)-fixing micro-organisms. The highest (15)N enrichments were consistently observed in the roots, although the total quantity of (15)N incorporated into the soil was much greater. The rate of (15)N incorporation into roots was much higher at the heading than at the tillering stage of growth. Definite enrichments were also found in the basal node and in the lower outer leaf sheath fractions after 3 days of exposure at the heading stage. Thirteen days was the shortest time period in which definite (15)N enrichment was observed in the leaves and panicle. When plants were exposed to (15)N(2) for 13 days just before heading and then allowed to mature in a normal atmosphere, 11.3% of the total (15)N in the system was found in the panicles, 2.3% in the roots, and 80.7% in the subsurface soil. These results provide direct evidence of heterotrophic N(2) fixation associated with rice roots and the flooded soil and demonstrate that part of the newly fixed N is available to the plant.

An arginyl-transfer ribonucleic Acid protein transferase from cereal embryos.

Embryos from rice (Oryza sativa L. var. Bluebonnet) and wheat (Triticum aestivum L.) contain an aminoacyl-tRNA protein transferase which transfers arginine from arginyl-tRNA to the N terminus of a protein acceptor. The activity was measured in vitro in a reaction mixture containing embryo supernatant fraction, buffer, sulfhydryl reagent, and arginyl-tRNA. It was not dependent on the usual cofactors for ribosomal protein synthesis, nor was it sensitive to cycloheximide or puromycin. However, the activity was inhibited by ribonuclease. The enzyme was purified 33-fold from a crude homogenate of rice embryos. An apparent endogenous substrate from rice embryos was prepared free of transferase activity; however, the transferase was not purified sufficiently to show absolute dependence on the presence of this endogenous substrate.

Dissociation of ribosomes and seed germination.

Ribosomes from rice embryos (Oryza sativa) were dissociated into ribosomal subunits in vitro by systematic reduction of the Mg(2+) concentration. Ribosomes from imbibed (28 C) embryos were more easily dissociated than those from nonimbibed embryos. This was not observed with ribosomes from either imbibed, nonviable embryos, or from embryos imbibed at 0 C. Ribosomes from embryos which had been imbided and subsequently dehydrated resembled ribosomes from nonimbibed embryos in their resistance to dissociation. The change in the resistance to dissociation was essentially complete after the first 20 minutes of imbibition at 28 C, and accompanied activation in vivo of protein synthesis as determined by amino acid incorporation in vitro. Ribosomes from either imbibed or nonimbibed embryos could be dissociated into subunits by 0.5 m KCl. These subunits were separated by density gradient centrifugation, and, if recombined, were active for polyphenylalanine synthesis in vitro. The individual subunits prepared from nonimbibed embryos could be replaced by the corresponding subunit fraction from imbibed embryos without loss of capacity to support polyphenylalanine synthesis. The change in the ease of dissociation of ribosomes appears to be a physiological process, and its possible relationship to the initiation of protein synthesis during seed germination is discussed.

Involvement of Aminoacyl-tRNA Transfer Factors in Polyphenylalanine Synthesis by Rice Embryo Ribosomes.

Two distinct heat labile factors (I and II) were found to be required for the in vitro synthesis of polyphenylalanine by rice ribosomes (Oryza sativa var. Bluebonnct) and polyuridylic acid. These factors were present in both the crude supernatant and on crude ribosomes. Factor I was removed from the crude ribosomes by [ill] (0.6%), while factor II was eliminated from deoxycholate washed ribosomes by a 0.5 m KCl wash.Factor I was purified from crude supernatant by calcium phosphate absorption and elution followed by polyacrylamide gel electrophoresis. Purified factor I was able to substitute for crude supernatant in supporting polyphenylalanine synthesis with deoxycholate washed ribosomes.The supernatant fraction from a KCl wash of deoxycholate ribosomes was found to contain factor II but no factor I activity. Factor II activity was also present in the calcium elutant fraction prepared from crude supernatant. Both factors I and II were required in order to substitute for crude supernatant in supporting polyphenylalanine synthesis with KCl washed ribosomes.The optimum Mg concentration for polyphenylalanine synthesis with d-oxycholate ribosomes was 4.5 mm with crude supernatant, and 6.5 mm with purified factor I. However, with KCl ribosomes supplemented with factors I and II, the optimum was 8.5 mm.Analytical ultracentrifugation Schlieren patterns indicated that KCl washed ribosomes contained a significant quantity of ribosomal subunits.

A soluble fraction requirement in the transfer reaction of protein synthesis by rice embryo ribosomes.

The requirements for the transfer of (14)C-phenylalanine from yeast soluble ribonucleic acid to protein in vitro by rice (Oryza sativa L. var. Bluebonnet) ribosomes have been investigated. An absolute requirement for polyuridylic acid, 2-mercaptoethanol, guanosine triphosphate, magnesium, and potassium or ammonium ions and ribosomes has been demonstrated. Ribosomes washed in 0.5% sodium deoxycholate also required the presence of rice supernatant. The optimum concentration of magnesium ion for the reaction was approximately 7 mm, while 60 mm of either ammonium or potassium ion gave maximum transfer of phenylalanine in this heterologous system. The optimum concentration of guanosine triphosphate required varied with the presence or absence of the phosphoenolpyruvate-pyruvate kinase generating system. Without the system, the optimum concentration was 1.5 mm, but in its presence the optimum was approximately 0.1 mm.