TY - JOUR A1 - Gomez-Merino, Fernando Carlos A1 - Arana-Ceballos, Fernando Alberto A1 - Trejo-Tellez, L. I. A1 - Skirycz, Aleksandra A1 - Brearley, C. A. A1 - Dormann, P. A1 - Müller-Röber, Bernd T1 - Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration : the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development N2 - Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at similar to 80 mu M inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development Y1 - 2005 SN - 0021-9258 ER - TY - JOUR A1 - Lin, W. H. A1 - Wang, Y. A1 - Müller-Röber, Bernd A1 - Brearley, C. A. A1 - Xu, Z. H. A1 - Xue, H. W. T1 - At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis N2 - Phosphatidylinositol signaling pathway and the relevant metabolites are known to be critical to the modulation of different aspects of plant growth, development, and stress responses. Inositol polyphosphate 5-phosphatase is a key enzyme involved in phosphatidylinositol metabolism and is encoded by an At5PTase gene family in Arabidopsis thaliana. A previous study shows that At5PTase11 mediates cotyledon vascular development probably through the regulation of intracellular calcium levels. In this study, we provide evidence that At5PTase13 modulates the development of cotyledon veins through its regulation of auxin homeostasis. A T-DNA insertional knockout mutant, At5pt13-1, showed a defect in development of the cotyledon vein, which was rescued completely by exogenous auxin and in part by brassinolide, a steroid hormone. Furthermore, the mutant had reduced auxin content and altered auxin accumulation in seedlings revealed by the DR5:beta-glucuronidase fusion construct in seedlings. In addition, microarray analysis shows that the transcription of key genes responsible for auxin biosynthesis and transport was altered in At5pt13-1. The At5pt13-1 mutant was also less sensitive to auxin inhibition of root elongation. These results suggest that At5PTase13 regulates the homeostasis of auxin, a key hormone controlling vascular development in plants Y1 - 2005 SN - 0032-0889 ER - TY - JOUR A1 - Xu, J. A1 - Brearley, C. A. A1 - Lin, W. H. A1 - Wang, Y. A1 - Ye, R. A1 - Müller-Röber, Bernd A1 - Xu, Z. H. A1 - Xue, H. W. T1 - A role of Arabidopsis inositol polyphosphate kinase, AtIPK2 alpha, in pollen germination and root growth N2 - Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca2+ concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca2+ concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores Y1 - 2005 SN - 0032-0889 ER - TY - JOUR A1 - Gomez-Merino, Fernando Carlos A1 - Brearley, C. A. A1 - Ornatowska, Magdalena A1 - Abdel-Haliem, Mahmoud E. F. A1 - Zanor, Maria Ines A1 - Müller-Röber, Bernd T1 - AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn- glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression N2 - Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode similar to55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K-m and V-max values were 125 muM for DAG and 0.25 pmol of PA min(-1) mug(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg2+-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca2+. AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2- dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4degreesC), pointing to a role in cold signal transduction Y1 - 2004 SN - 0021-9258 ER -