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Articles

Nitrogen Metabolism in Soybean Tissue Culture

II. Urea Utilization and Urease Synthesis Require NI²⁺

¹ Department of Genetics, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, Connecticut 06504

Potassium citrate (10 mM, pH 6) inhibits the growth of cultured (Glycine max L.) cells when urea is the sole nitrogen source. Ureadependent citrate toxicity is overcome by three separate additions to the growth medium: (a) NH₄Cl (20 mM); (b) high levels of MgCl₂ (10 mM) or CaCl₂ (5-10 mM); (c) low levels of NiSO₄ (10^–2 mM). Additions of 10^–2 mM NiSO₄ not only overcome citrate growth inhibition but the resultant growth is usually better than urea-supported growth in basal medium (neither added citrate nor added nickel). In the absence of added citrate, exceedingly low levels of NiSO₄ (10^–4 mM) strongly stimulate urea-supported growth in suspension cultures.

Citrate does not inhibit growth when arginine is sole nitrogen source. However, cells using arginine have no net urease synthesis in the presence of 10 mM potassium citrate. When 10^–2 mM NiSO₄ is added to this medium, urease specific activity is 10 times that observed in basal medium lacking both citrate and added nickel.

Citrate is a chelator of divalent cations. That additional Mg²⁺ or Ca²⁺ alleviates urea-dependent citrate toxicity indicates that citrate is acting by chelation, probably of another trace divalent cation; this is probably Ni²⁺ since at 10^–2 mM it overcomes citrate toxicity and at 10^–4 mM it stimulates urea-supported growth in the absence of citrate. That ammonia overcomes citrate toxicity indicates that the trace Ni²⁺ is essential specifically for the conversion of urea to ammonia. Ni²⁺ stimulation of urease levels in arginine-grown cells supports this contention.

In basal medium, soybean cells grow slowly with urea nitrogen source presumably because the trace amounts of Ni²⁺ present (10^–6 mM) are growth-limiting.

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