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5NJNMR Determination of Asparagine and Glutamine Nitrogen

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5NJNMR Determination of Asparagine and Glutamine Nitrogen Plant Physiol. (1982) 69, 308-313 0032-0889/82/69/0308/06/$00.50/0 ['5NJNMR Determination of Asparagine and Glutamine Nitrogen Utilization for Synthesis of Storage Protein in Developing Cotyledons of Soybean in Culture Received for publication May 26, 1981 and in revised form September 2, 1981 THOMAS A. SKOKUT', JOSEPH E. VARNER, JACOB SCHAEFER, EDWARD 0. STEJSKAL, AND ROBERT A. MCKAY Department ofBiology, Washington University, St. Louis, Missouri 63130 (T. A. S., J. E. V.) and Physical Sciences Center, Monsanto Co., St. Louis, Missouri 63166 (J. S., E. 0. S., R. A. McK.) ABSTRACT Although asparagine and glutamine are similar in chemical structure, the metabolic pathways responsible for transfer of their Solid-state I'5NINMR was used to measure the use of the amide and nitrogen to other compounds are basically different. The amide amino nitrogens of glutamine and asparagine for synthesis of storage nitrogen of glutamine can be directly transferred to a-ketoglutar- protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No ate to form glutamate by the action of glutamate synthase (13). If major discrimination in the incorporation of the amide or amino nitrogens the amide nitrogen of asparagine is to be transferred to other of glutamine into protein is apparent, but the same nitrogens of asparagine amino acids, the asparagine molecule first must be hydrolyzed to are used with a degree of specificity. During the first seven days in culture ammonium and aspartate by the enzyme asparaginase; an enzyme with asparagine as the sole nitrogen source, the amino nitrogen donates system similar to glutamate synthase that could transfer asparagine approximately twice as much nitrogen to protein as does the anmde amide nitrogen to a-ketoglutarate or glutamate has not been nitrogen. The use of the amide nitrogen increases with longer periods of detected in higher plants (7). The free ammonium is reassimilated culture. The reduced use of the amide nitrogen was confirmed by its early to form glutamine via glutamine synthetase (13). These two path- appearance as ammonium in the culture medium. The amide nitrogen of ways of utilization of amide nitrogen are believed to be operating asparagine was found at aDl times to be an essential precursor for protein during seed development because potassium-dependent asparagi- because of its appearance in protein in residues whose nitrogens were not nase activity has been detected in the developing seeds of a supplied by the amino nitrogen. In addition, methionine sulfoximine in- number of legumes (23), and glutamate synthase activity has been hibited growth completely on asparagine, indicating that some ammonium detected in developing cotyledons of soybean and pea (24, 25). assimilation is essential for storage protein synthesis. These results indi- The amino nitrogens of glutamine and asparagine can both be cate that in a developing cotyledon, a transaminase reaction is of major transferred to other amino acids by the action of transaminases importance in the utilization of asparagine for synthesis of storage protein (29) using the glutamate and aspartate formed as a result of the and that, at least in the early stages of cotyledon development, reduced reactions described above. However, the amino nitrogen of aspar- activities of ammonium-assimilating enzymes in the cotyledon tissue or in agine can also be transferred directly to pyruvate, glyoxylate, ox- other tissues of the seed or pod may be a limiting factor in the use of aloacetate, or a-ketoglutarate by the action of asparagine trans- asparagine-amide nitrogen. aminase (7). Asparagine transaminase activity has been found in leaves of lupin, soybean, and pea (8, 10, 27) and thus might be present in developing seeds. In this paper we report the use of '5N-labeled asparagine and glutamine, and solid-state magic-angle cross-polarization [15N]- NMR (19, 20) to study the metabolism of these compounds in developing cotyledons of soybean in culture. The culture method The production of storage protein in a developing embryo is we use, Thompson et al. (28), has been shown to be an appropriate dependent upon the flow of nitrogen compounds to the immature system for study of legume seed storage protein synthesis under seed from other parts of the plant and the subsequent transfer of controlled conditions. The NMR analysis can be performed on nitrogen from these compounds to the amino acids required for intact cotyledons thereby avoiding elaborate digestion, separation, protein synthesis. Although ureides are found in substantial quan- derivitization, and purification procedures normally required in tities in the translocation stream of many legumes (12, 18), aspar- stable isotope studies. From our labeling and NMR experiments, agine and glutamine are usually the major amino acids present we have determined the extent to which the amide and amino (16, 17). In soybean, asparagine can represent as much as 60% of nitrogens of these amino acids contribute to the synthesis of the total amino acid nitrogen extracted from stem exudate (26), storage protein. We find that the amide and amino nitrogens of suggesting that it is an important nitrogen source for protein glutamine are used similarly in protein synthesis, whereas the synthesis in developing seeds. Glutamine plays a central role in corresponding nitrogens of are not. the assimilation of ammonium in plants (13) and has been found asparagine to support substantial growth of soybean cotyledons in culture (28), indicating that it, too, may play an important role in synthesis MATERIALS AND METHODS of storage protein. Growth of Plants. Glycine max (cv. Elf) were grown in chambers under conditions previously described (21). At the time of plant- 'Present address: Monsanto Agricultural Products Co., 800 N. Lind- ing, seeds were inoculated with Rhizobium japonicum. After the bergh Boulevard, St. Louis, MO 63166. appearance of the first trifoliolate, the plants were fertilized three 308 PROTEIN SYNTHESIS IN SOYBEAN COTYLEDONS 309 times a week with Hoagland solution. Between 70 and 90 days above reagents was subtracted from all values. The concentrations after planting, selected pods were removed and the immature of ammonium in the unknown samples were calculated from a seeds were used to initiate organ cultures of cotyledons. standard absorbance curve obtained with known concentrations Growth of Cotyledons in Culture. Immature cotyledons were of NH4Cl in 0.01 N HCI. grown in culture using a slightly modified procedure ofThompson Ammonium content of the cotyledon tissue was obtained from et al. (28). The excised cotyledons were rinsed with sterile water, the 80% ethanol extract. The extract was acidified with HCI to pH blotted dry, and transferred to a culture flask which contained 2.0 and evaporated to dryness. The dried residue was redissolved liquid medium. Each flask was weighed before and after adding in H20 and a portion was subjected to the above ammonium assay the cotyledon to obtain an initial fresh weight. The initial fresh procedure. weight of each cotyledon ranged between 5 and 20 mg. Stable Isotopes. '5N-labeled amino acids (95-98 atom % 15N) The culture medium used was that described by Thompson et were obtained from Merck (Montreal, Canada) and Stohler Iso- al. (28), with the only difference being the concentration of the tope Chemicals (Azusa, CA). The ['5N]amide asparagine used in nitrogen source. The medium was prepared without the amino these experiments was also labeled with 13C at the number 4 acid nitrogen source and adjusted to pH 6.0 with NaOH. Ten ml carbon only (90 atom % 13C). of medium, in 50-ml screw-top Erlenmeyer flasks, were sterilized [15NINMR. Magic-angle [15N]NMR spectra were obtained at by autoclaving. The amino acid nitrogen source was prepared as 9.12 MHz using matched spin-lock cross-polarization transfers a stock solution, adjusted to pH 6.0 with NaOH, and sterilized by with 1-ms single radio frequency contacts and 25 kHz His with filtration with an Acrodisc filter (0.2 iLm pore size; Gelman, Ann the dried samples contained in a Beams-Andrew 420-!L hollow Arbor, MI). Two ml of the sterile amino acid stock solution were rotor spinning at 1.5 kHz (19). Technical details of the spinning added to each culture flask containing sterile medium so that a and cross-polarization procedures have been reported elsewhere final concentration of 30 mm ~lutamine or 60 mm asparagine ('5N- (20). Fast cross-polarization rates for protonated nitrogens, long labeled or natural abundance 5N depending upon the experiment) proton rotating-frame lifetimes, and high concentrations of pro- was obtained. tons in these biological samples ensure representative NMR inten- The culture flasks containing one cotyledon each were incu- sities for all nitrogens (19, 20). The one exception is nitrogen in bated for various times at 28 ± 1 °C in an incubator shaker (Model the form of ammonia or ammonium ion, where internal molecular G-25R; New Brunswick Scientific, Edison, NJ). The flasks were motion decreases the cross-polarization transfer rates, resulting in shaken with a rotary motion describing a 1-inch circle at 100 rpm signal intensities which underestimate the ammonium nitrogen and were illuminated from above with a 20-w cool white fluores- present. The degree of the underestimate can be evaluated by a cent bulb (GE) at a distance of 30 cm. measurement of cross-polarization transfer from protons to nitro- Preparation of Cotyledons for NMR Analysis. After incubation, gens as a function of the time during which the two spin systems the cotyledons were rinsed with distilled H20, blotted, weighed, are in radio frequency contact (20). frozen in liquid N2, and lyophilized. After lyophilization, the The amount of 1 N present in the protein of the ethanol- cotyledons were subjected to [ 5N]NMR analysis. When only the extracted cotyledons was calculated from the NMR spectra, using nitrogen present in protein was to be observed, the cotyledons a natural abundance cross-polarization ['5N]NMR spectrum of a were extracted with 80% ethanol to remove free amino acids.
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