Nitrate Reductase- and Nitric Oxide-Dependent Activation of Sinapoylglucose:malate sinapoyltransferase in Leaves of Arabidopsis thaliana

Nitrate reductase (NR) activity is necessary for the synthesis of nitric oxide (NO), a key signaling molecule in plants. Here, we investigated the effect of NR deficiency on NO production and phenylpropanoid metabolism of Arabidopsis thaliana leaves. HPLC-mass spectrometry analysis showed that the NR double mutant (nia1 nia2) is deficient in the synthesis of sinapoylmalate (SM), the main phenylpropanoid end-product in wild-type leaves, resulting in accumulation of its precursor sinapoylglucose (SG). While real-time PCR analysis revealed no significant difference at the transcript level, sinapoylglucose:malate sinapoyltransferase (SMT) activity in leaf extracts was reduced in the mutant compared with the wild type. The low levels of SM in nia1 nia2 leaves do not result from the deficient nitrogen incorporation into amino acids, since the recovery of the amino acid content of nia1 nia2 by irrigating the plants with glutamine did not change the metabolic profile of this mutant. In contrast, an increased supply of nitrate stimulated NR activity and NO production, and enhanced SM and decreased SG levels in both genotypes. Nevertheless, sinapic acid esters in nia1 nia2 were not recovered when compared with those detected in the leaves of the wild-type plant. Mutant plants grown in medium supplemented with malate and an NO donor recovered SM to the levels of wild-type leaves. Overall, the results suggest that SMT activity is dependent on the NR-dependent steady-state levels of NO during plant development.