Effect of Methionine Sulfoximine on the Induction of Nitrate Reductase and Metabolic Nitrate Pool Size in Barley Roots

Abstract

The effect of methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, on the induction of nitrate reductase activity (NRA) and metabolic NO₃ ^- pool size in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activity was induced at different NO₃ ^- concentrations (0 to 100 mM) in the presence or absence of 0.25 mM MSO and assayed in vitro. Metabolic NO ⁻₃ pool concentration was determined using the minus NO ⁻₃ in vivo assay. In the absence of MSO, induction of NRA plateaued at 0.1 mM external NO ⁻₃ , even though total NO ⁻₃ concentration in roots increased with increasing external NO ⁻₃ . Treatment with MSO enhanced the induction of NRA above 0.1 mM external NO ⁻₃ . Thus, in roots supplied with MSO at 100 mM NO₃ ^-, the level of the enzyme activity was 70 to 80% higher than in roots not supplied with MSO, even though total NO ⁻₃ concentration in MSO-treated roots was 20 to 30% lower. Induction of NRA also plateaued after 6 h of exposure to 100 mM NO ⁻₃ ; whereas in the presence of MSO, NRA continued to increase up to 24 h. Glutamine, supplied exogenously or accumulated internally by inhibiting its assimilation, had no effect on the induction of NRA. Metabolic NO ⁻₃ concentration in roots treated with MSO at 100 mM NO ⁻₃ was higher than in those not treated with MSO. In contrast, MSO had no effect on metabolic NO ⁻₃ concentration in roots induced with 0.1mM NO ⁻₃ . Metabolic NO ⁻₃ concentration remained constant between the 6 and 24 h induction period. However, MSO treatment almost doubled the metabolic NO ⁻₃ concentration during the same period. The results indicate that the metabolic NO ⁻₃ pool is saturated at 0.1 mM exogenous NO ⁻₃ and the enhancement of NRA by MSO induced with NO ⁻₃ concentrations greater than 0.1 mM may be due to increased availability of NO ⁻₃ in the metabolic pool.