Nitrate Reductase in Maize Roots: Localization and Molecular Characterization

Abstract

<p>Nitrate reductase (NR) is the first enzyme involved in the pathway of nitrate assimilation in plants. It converts nitrate to nitrite. By including the serine protease inhibitor, chymostatin, in the extraction buffer, NR from maize (Zea mays L.) roots was stabilized in vitro. Contrary to early results, it was found in substantial amounts in the mature regions of the root. Two isozymes of NR were identified, an NADH monospecific form found predominantly in the root tip, and an NAD(P)H bispecific form which was predominant in the mature portion of the roots. Both isozymes were found to reach substantial levels of activity, approximately one-third to one-half the levels found in shoots. The levels of NR activity in both shoots and roots varied with the age of the plants and the conditions of growth. Subsequent purification and biochemical characterization of the two isozymes suggested similarities in the characteristics of the isozymes. However, the NADH form had an exceptionally high Km for NADH which suggests that the NADH:NR may not be active in the assimilatior. of nitrate as it may not be able to compete with other dehydrogenases for reductant.</p> <p>A partial cDNA clone of root NR was isolated, sequenced and identified as a gene distinct from the gene which codes for NR in maize leaves. The NR in maize roots was affected in a positive manner by nitrate at the levels of activity and transcription. However, it did not appear to be affected by either a diurnal rhythm or directly by light, as was found for leaf NR. Through the use of the tissue print hybridization technique, NR mRNA was found to be expressed throughout the maize root with the exception of the root tip.</p> <p>In conclusion, NR in maize roots is present in high enough amounts to account for a substantial level of nitrate reduction in the roots, suggesting that maize roots have an important role in the overall metabolism of nitrate in maize.</p>