Nitrate reductase suppression by light: Protein abundance vs. post-translational modification

Plants typically acquire soil nitrogen in the form of nitrate, which is enzymatically reduced prior to assimilation. The initial reduction of nitrate to nitrite is mediated by nitrate reductase (NR). NR activity is regulated transcriptionally and post-translationally. Post-translational regulation is modulated by reversible phosphorylation; the inhibitory complex is formed by the association of magnesium ions and 14-3-3 proteins with the phosphorylated NR protein. Nitrate and light have been shown to modify NR activity, typically increasing NR activity. Current research has demonstrated that NR activity in tissues of ivy leaf morning glory (Ipomoea hederacea) and radish (Raphanus sativus) seedlings can be suppressed by light. The underlying mechanism of NR activity suppression has yet to be determined. Previous experiments used in vivo activity measurements of intact tissues. In vivo activity measurements quantify the product, nitrite, but cannot determine NR protein levels or reveal post-translational modifications of the NR protein. The application of an in vitro assay to tissue extracts will permit a corroborative measurement to confirm the suppression of NR activity in light-treated tissues. Further, in vitro assays allow for manipulation of the reaction environment, specifically sequestration of divalent magnesium, providing data surrounding the phosphorylation state of the enzyme. The inclusion of a chelating agent in the in vitro assay should provide evidence for NR protein phosphorylation state; increased activity in the presence of a chelating agent, relative to lower activity in its absence, would suggest that inhibition is due to the formation of the magnesium-containing inhibitory complex by the phosphorylated form