Cloning and characterization of NAD+and NADH kinases from the model higher plant, Arabidopsis thaliana.

摘要

Three nicotinamide adenine dinucleotide kinase isoforms (NADKs, designated NADK1, NADK2, and NADK3 respectively) were identified in the model higher plant Arabidopsis thaliana's genome and examined at the biochemical, molecular, subcellular, and whole-plant level. NADK1 and NADK2 were found to be NAD+-specific kinases while NADK3 was found to be a novel plant NADH-specific kinase. Recombinant NADK1 and NADK3 were found not to interact with calmodulin (CaM) while native and recombinant NADK2 was found to bind CaM by virtue of its extended N-terminal domain containing a CaM-binding domain. NADK1 was found to be a cytosolic enzyme while NADK2 and NADK3 were localized exclusively to the chloroplast stroma and peroxisomal matrix, respectively. NADK2 activity remained CaM-insensitive when the chloroplast transit peptide was removed but it was found to be activated by simulated stromal light conditions. NADKs were found to differ in their tissue expression patterns throughout development based upon NADK promoter::GUS (beta-glucuronidase) reporter assays. NADK1::GUS was strongly expressed in the root and weakly in the shoot tissue, with minor expression in reproductive tissue. NADK2::GUS expression was strongest in photosynthetic tissue but was also found in roots and reproductive tissue. Strong NADK3::GUS expression was found to be restricted to vascular and reproductive tissue. Therefore, Arabidopsis NADKs exhibit different properties with respect to activity, compartmentalization, and expression. Since NADKs are the sole means of de novo NADP(H) biosynthesis and many enzymes and processes depend on NAD(P), NADKs are concluded to have distinctive roles in plant metabolism and stress response.