A ¹⁴N and ¹⁵N Nuclear Magnetic Resonance Study of Nitrogen Metabolism in Shoot-Forming Cultures of White Spruce (Picea glauca) Buds

Abstract

Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other aNH2 groups), Pro, Ala, and the side chain groups in Gin, Arg, Om, and 'y-aminobutyric acid could be detected in in vivo 15N NMR spectra. Peaks for aamino groups, Pro, N03-and NH4 could also be identified in 14N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that 15N-labeled NH4 and N03-are first incorporated into the amide group of Gin and then in the aNH2 pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH4+ is much faster than that of N03-. Consequently after 10 days of growth more than 70% of the newly synthesized intemal free amino acid pool derives its nitrogen from NH4+ rather than N03-. If NH4+ is omitted from the medium, no N03-is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH4 and N03-are both required for the induction of nitrate-and nitrite reductase.The use of tissue culture technology in the micropropagation of plants is the widest application of this technology to date (5, 26). The process of de novo organ formation in tissue cultures can be manipulated by the judicious choice of the explant, medium and culture environment. The medium manipulation includes the selection of a proper mineral salt formulation, a key component of which is the concentration of inorganic nitrogen and the ratio of NO3-to NH4' (9). Differences in nitrogen assimilation and amino acid metabolism may lead to a change in the content and spectrum of amino acids and both structural and enzymatic proteins in

Nitrogen Metabolism in Lignifying Pinus taeda Cell Cultures

“…Nitrogen Metabolism in P. taedaviously assigned in nitrogen metabolism studies with white spruce (Picea glauca) buds (24). Importantly, no resonances at any stage corresponding to 15 NH 4 ϩ were observed, in accordance with earlier observations, such as with potato (Solanum tuberosum) slices, where it did not reach detectable levels (7) during active phenylpropanoid-glutamine synthetase/glutamate synthase metabolism.…”

“…Thus, both isotopic enrichment and total amounts (micrograms/g fresh weight) of each principal metabolite (Gln and Glu) were determined, following incubation of 15 N-Phe (99.9 atom %, 10 mM) with P. taeda cell cultures, for periods up to 96 h. As can be seen, the phenylalanine present in the soluble pool in the cells was ϳ90 atom % 15 N-enriched at all intervals sampled (24,48,72, and 96 h). But its amount decreased from ϳ677 g/gfw (t ϭ 24 h) to ϳ25 g/gfw (t ϭ 96 h) as a result of the utilization of its carbon skeleton for phenylpropanoid metabolism.…”

“…N-Glu were administered at a final concentration of 10 mM at t ϭ 0 h. For inhibitor studies, L-AOPP was administered at a final concentration of 0.1 mM at t ϭ 0 h, whereas MSO and AZA were at 5 mM. In each experiment, the cells were incubated at 25°C, over a time course of 24 N 2 ), and stored at Ϫ80°C until needed. Frozen cells were ground in a chilled mortar, extracted with cold EtOH (5 ml), with the resulting slurry transferred, by means of two rinses (3 ml of 95% EtOH each) into a conical tube.…”

The Role of Glutamate Dehydrogenase in Plant Nitrogen Metabolism

“…['5N]ammonium incorporation was analyzed using in vivo NMR and GC-MS. Figure 1 Figure 1 is similar to that observed previously by in vivo 5N NMR in the ectomycorrhizal fungus Cenococcum graniforme (12) and shoot-forming cultures of white spruce buds (27 In addition to preventing the incorporation of '5N into amino acids, MSO also affected the concentration of ammonium in the medium (Table III). In control cells the ammonium concentration dropped from 1.6 mm to 0 over 12 h, representing an uptake rate of 1.3 ,umol h-'g fresh wt-'.…”

In Situ Nuclear Magnetic Resonance of 15N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

“…of pulse labels under aerobic, dark conditions were reported for spruce buds (16) and cell suspension cultures of carrots (12). The aim of the present work was to develop further the in vivo "5N NMR studies by following the assimilation of ammonia in photoautotrophic microorganisms under photosynthetic conditions.…”