Effects of nitrate-induced promotion of de novo NAD synthesis in Arabidopsis thaliana

In higher plants, NAD synthesis starts with the oxidation of aspartate. This oxidation is catalyzed by aspartate oxidase (AO) encoded by a nitrate-inducible gene. To assess the effects of this upregulation of AO gene in response to a nitrate supply, a transgenic line was generated by introduction of AO gene under the control of a mutated AO promoter into the ao knockout mutant. Since, unlike the wild-type AO promoter, this mutated AO promoter was not activated by a nitrate supply, gene expression in this transgenic line was compared with that in another transgenic line that was generated by introduction of AO gene under the regulation of the wild-type AO promoter into the ao knockout mutant. Changes in gene expression in response to a nitrate supply were analyzed in these transgenic lines. Effects of nitrate-inducibility of AO gene on gene expression in Arabidopsis thaliana was assessed using transgenic lines expressing AO gene from the wild-type or nitrate-non-inducible mutated AO promoter. Hydroponically-grown transgenic plants (0d) were subjected to 3-day nitrogen-starvation treatment (Nstarvation3d) and then grown with 10 mM nitrate-containing solution for 24 hours (resupply).