Plant cells respond to ageing by implementing an emergency survival strategy: increase mitochondrial pyridine dinucleotides pool via NMN-AT

Ageing is characterized by a progressive deterioration of physiological functions and metabolic processes. The process of ageing correlates with a loss of antioxidant capacity and consequentily with an increase of ROS. Production of mitochondrial ROS by the electron transport chain is largely dependent on the NAD(P) status as well. The cellular redox state influences many metabolic, signalling and transcriptional processes in the cell. Such signalling pathways consume NAD(P) which is replenished via a combination of de novo and salvage pathways that contribute to the biosynthesis of NAD(P) in all organism. Both pathways converge at the transfer of nicotinamide mononucleotide (NMN) or its acid form NaMN on to the adenylyl group of ATP under pyrophosphatase release. The reaction is catalysed by NMN adenylyltransferase (EC 2.7.7.1) a member of the nucleotidyl transferase α/β-phosphodiesterase superfamily, that catalyzes the reversible reaction NMN+ ATP NAD+ + PPi. The biosynthesis of NADP requires the phosphorilation of NAD catalysed by NADK (ATP:NAD 2’-phosphotransferase) (EC 2.7.1.B1) NADK activity in plants, has been found in the cytoplasm, chloroplast and mitochondria. Thus NMN is an important metabolite for the maintenance of normal NAD(P) biosynthesis. Important information on the pathways involved in NAD(P) synthesis in plants is beginning to appear, but many outstanding questions remain. It is well know that a substantial fraction of the cellular NAD pool is compartmentalized within the mitochondria. To date, in PUBMED leterature, are not reported studies that deal with NAD synthesis in plant mitochondria. Structural, kinetic and regulatory properties of NMN-AT, from different sources, have been studied. NMNAT isozymes, encoded by different genes and showing distinctive organelle and tissue distribution, have been reported in eukaryotes, except for plants but searches of the TAIR database suggested that Arabidopsis thaliana contains only a single-copy NMN-AT gene: AtNMNAT (At5g55810). Recently our works (Di Martino, Pallotta. New insights into the NAD synthesis in mitochondria isolated from fresh and aged-dehydrated tubers of Jerusalem artichoke. IX National Congress FISV 2007; Pallotta, Di Martino Existence of intramitochondrial nicotinamide mononucleotide adenylyl-transferase activity, which allows for NAD+ synthesis from NMN and endogenous ATP in aged-dehydrated slices tubers of Helianthus tuberosus. Cell Stress & Chaperones 2007 Vol. 12, Issue 2 1355), demonstrated, for the first time, that plant mitochondria contain an own NMN-AT. The experimental strategy we wished to pursue was to induce aging in tubers of Helianthus tuberosus. The fact that Jerusalem artichokes tubers can be used as high fiber (i.e., inulin) food with no glucose and little fructose, has generated considerable interest in these plants as an alternative food source in diabetic patients, thus we have chosen these tubers as a model system for our research. We have exploited the opportunity to characterize how the mechanisms of ageing could change NAD(P) levels in plant mitochondria. NMN was added to mitochondria isolated from ageing slices tubers of Helianthus tuberosus and NAD(P) content was tested, in different experimental conditions, both via HPLC and spectroscopically. Our data indicate that the incresead NAD(P) pool, respect to fresh plant mitochondria, is due to an increased activity of almost one enzyme involved in NAD salvage, i.e. NMN-AT. The increased activity of NMN-AT provided compelling evidence that oxidative stress triggers changes in mitochondrial NAD(P) biosynthesis pathways. These results will be discussed in the context of current theories of ageing that have been documentated in according with plant and mammalian literature.