Nitrogen Metabolism in Soybean Tissue Culture

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Abstract
Cultured soybean (Glycine max cr. ''Kanrich'') cells grow with 25 mM urea as the sole N source but at a slower rate than with the Murashige and Skoog (MS) N source of 18.8 mM KNO3 and 20.6 mM NH4NO3. Growth with urea is restricted by 18.8 mM NO3-, 50 mM methylammonia, 10 mM citrate or 100 .mu.M hydroxyurea, substances which are much less restrictive or nonrestrictive in the presence of ammonia nitrogen source. The restrictive conditions of urea assimilation were examined as possible bases for selection schemes to recover urease-overproducing mutants. Since urease has higher methionine levels than the soybean seed proteins among which it is found, such selections may be a model for improving seed protein quality by plant cell culture techniques. Callus will not grow 1 mM urea plus 18.8 mM KNO3. Urease levels decrease 80% within 2 divisions after transfer from MS N source to 1 mM urea plus 18.8 mM KNO3. Hydroxyurea is a potent inhibitor of soybean urease, and this appears to be the basis for its inhibition of urea utilization by callus cells. Stationary phase suspension cultures grown with MS N source exhibit trace or zero urease levels. Soon after transfer to fresh medium (24 h after escape from lag), urease levels increase in the presence of both MS or urea N source. The increase is 10-20 times greater in the presence of urea. NH4Cl (50 mM) lowers urease induction by 50%, whereas 50 mM methylammonium chloride results in more drastic reductions in urea-stimulated urease levels. Citrate (10 mM) completely blocks urease synthesis in the presence of urea. Ammonia and methylammonia do not inhibit soybean urease nor do they appreciably inhibit urea uptake by suspension cultures. It appears likely that methylammonia inhibits urea utilization in cultured soybean cells primarily due to its repressive effect on urease synthesis. Citrate does not inhibit urease activity in vitro and exhibits only a partial inhibition (0-50% in several experiments) of urea uptake. It appears likely that the citrate elimination of urease production by cultured soybean cells is due to its chelation of trace Ni2+ in the growth medium. It was reported that jack bean (Canavalia ensiformis) urease contains N at the active site.