A novel Arabidopsis thaliana gene (AtNADK-1) was identified based on its response to radiation and oxidative stress. Levels of AtNADK-1 mRNA increase eight-fold following exposure to ionising radiation and are enhanced three-fold by treatment with hydrogen peroxide. The gene also appears to be differentially regulated during compatible and incompatible plant-pathogen interactions in response to Pseudomonas syringae pv. tomato. The full-length AtNADK-1 cDNA encodes a 58-kDa protein that shows high sequence homology to the recently defined family of NAD(H) kinases. Recombinant AtNADK-1 utilises ATP to phosphorylate both NAD and NADH, showing a two-fold preference for NADH. Using reverse genetics, we demonstrate that AtNADK-1 deficient plants display enhanced sensitivity to gamma irradiation and to paraquat-induced oxidative stress. Our results indicate that this novel NAD(H) kinase may contribute to the maintenance of redox status in Arabidopsis thaliana.

An Arabidopsis thaliana gene (AtLPP1) was isolated on the basis that it was transiently induced by ionizing radiation. The putative AtLPP1 gene product showed homology to the yeast and mammalian lipid phosphate phosphatase enzymes and possessed a phosphatase signature sequence motif. Heterologous expression and biochemical characterization of the AtLPP1 gene in yeast showed that it encoded an enzyme (AtLpp1p) that exhibited both diacylglycerol pyrophosphate phosphatase and phosphatidate phosphatase activities. Kinetic analysis indicated that diacylglycerol pyrophosphate was the preferred substrate for AtLpp1p in vitro. A second Arabidopsis gene (AtLPP2) was identified based on sequence homology to AtLPP1 that was also heterologously expressed in yeast. The AtLpp2p enzyme also utilized diacylglycerol pyrophosphate and phosphatidate but with no preference for either substrate. The AtLpp1p and AtLpp2p enzymes showed differences in their apparent affinities for diacylglycerol pyrophosphate and phosphatidate as well as other enzymological properties. Northern blot analyses showed that the AtLPP1 gene was preferentially expressed in leaves and roots, whereas the AtLPP2 gene was expressed in all tissues examined. AtLPP1, but not AtLPP2, was regulated in response to various stress conditions. The AtLPP1 gene was transiently induced by genotoxic stress (gamma ray or UV-B) and elicitor treatments with mastoparan and harpin. The regulation of the AtLPP1 gene in response to stress was consistent with the hypothesis that its encoded lipid phosphate phosphatase enzyme may attenuate the signaling functions of phosphatidate and/or diacylglycerol pyrophosphate that form in response to stress in plants.Phospholipids are major structural components of biological membranes in plants, animals, and yeast. These molecules also serve as a reservoir for several lipid-signaling molecules. PA 1 and DG are intermediates in the biosynthesis of phospholipids and triacylglycerols (1, 2). PA is an intermediate for the synthesis of the major phospholipids, which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol. The dephosphorylation of PA to DG allows for the subsequent production of phosphatidylcholine and triacylglycerol, the major components of eukaryotic membranes and storage lipid, respectively (1, 2). In addition, PA, DG, and DGPP are products of lipid metabolism that serve roles as second messengers in several cellular signal transduction pathways (3-8). The regulation of these cell-signaling pathways may be achieved, at least in part, by lipid phosphate phosphatase enzymes that catalyze the sequential conversion of DGPP to PA and of PA to DG (5, 7, 9).Recent studies with a variety of plant systems have shown that PA and DGPP transiently accumulate after elicitor treatment or in response to stress (5, 10 -12). These observations have suggested that these phospholipid molecules play a role in plant signal transduction (10, 11). Lipid second messengers in plant cell s...

Screening for mRNAs that accumulate after DNA damage induced by ionizing radiation, we have isolated a 2.0-kb cDNA coding for a new Arabidopsis PEST-box protein named AtGR1 (A. thaliana gamma response 1) with an expression profile similar to that observed for several plant cell cycle-related proteins. Using an anti-AtGR1 antibody, we have shown that the AtGR1 protein is expressed at basal levels in mitotically dividing cells (meristematic tissues and organ primordia) and at a strongly enhanced level in endoreduplicating cells (stipules, trichomes). Using transgenic Arabidopsis plants that express the GUS reporter gene under the control of the AtGR1 promoter, we have demonstrated that the observed AtGR1 protein distribution is due to the promoter activity. Our results suggest that basal AtGR1 levels are associated with progression through mitosis, whereas elevated intracellular levels of AtGR1 seem to induce changes between the S and M phases of the cell cycle that trigger somatic cells to enter the endoreduplication cycle. Ionizing radiation-induced rapid and dose-dependent accumulation of AtGR1 mRNA in cell cultures and plant tissues leads to tissue-specific accumulation of AtGR1 protein, best observed in ovules, which never undergo an endoreduplication cycle. It therefore appears that the radiation-induced transient AtGR1 accumulation reflects DNA damage-dependent transient cell cycle arrest before mitosis, which is necessary to accomplish DNA repair prior to chromosome segregation and cytokinesis.