ABSTRACT
Expression of ZmCPK11, a member of the maize (Zea mays) calcium-dependent protein kinases (CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56-kDa CDPK has been observed in damaged leaves. In the present work, it is shown that the 56-kDa CDPK, identified as ZmCPK11, is also activated in non-wounded leaves as an element of systemic wound response. Moreover, an increase of the enzyme's activity and induction of ZmCPK11 expression was observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling, transgenic Arabidopsis thaliana plants in which c-Myc-ZmCPK11 was expressed under control of the CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c-Myc-ZmCPK11 was activated upon wounding and touching. Furthermore, pre-treatment with acetylsalicylic acid (acSA), an inhibitor of jasmonic acid (JA)-dependent wound signaling, abolished the wound-induced activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of ZmCPK11 and other wound-responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1 (ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level by LA and MeJA, is a component of touch- and wound-induced pathway(s), participating in early stages of local and systemic responses.
Subject(s)
Cyclopentanes/metabolism , Mechanotransduction, Cellular/physiology , Oxylipins/metabolism , Plant Physiological Phenomena/physiology , Protein Kinases/metabolism , Zea mays/enzymology , Adaptation, Physiological , Arabidopsis/genetics , Arabidopsis/metabolism , Enzyme Activation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genes, Plant , Mechanotransduction, Cellular/genetics , Plant Growth Regulators/genetics , Plant Growth Regulators/metabolism , Plant Leaves/enzymology , Plants, Genetically Modified/metabolism , Protein Kinases/genetics , Stress, Physiological , Zea mays/geneticsABSTRACT
Asymmetrical diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) hydrolases are key enzymes controlling the in vivo concentration of Ap(4)A--an important signaling molecule involved in regulation of DNA replication and repair, signaling in stress response and apoptosis. Sequence homologies indicate that the genome of the model plant Arabidopsis thaliana contains at least three open reading frames encoding presumptive Ap(4)A hydrolases: At1g30110, At3g10620, and At5g06340. In this work we present efficient overexpression and detailed biochemical characteristics of the AtNUDX25 protein encoded by the At1g30110 gene. Aided by the determination of the binding constants of Mn(Ap(4)A) and Mg(Ap(4)A) complexes using isothermal titration calorimetry (ITC) we show that AtNUDX25 preferentially hydrolyzes Ap(4)A in the form of a Mn(2+) complex.
Subject(s)
Acid Anhydride Hydrolases/chemistry , Arabidopsis/enzymology , Ions , Manganese/chemistry , Adenosine Diphosphate Ribose/chemistry , Amino Acid Sequence , Calorimetry/methods , DNA Repair , DNA Replication , Molecular Sequence Data , Open Reading Frames , Pyrophosphatases/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Spectrometry, Mass, Electrospray Ionization/methods , Nudix HydrolasesABSTRACT
A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.
Subject(s)
Arabidopsis/enzymology , Insecta/genetics , Pyruvate Dehydrogenase Complex/metabolism , Animals , Catalysis , Cell Line , Cloning, Molecular , Cytoplasm/enzymology , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genetic Vectors/genetics , Insecta/cytology , Mitochondria/enzymology , Protein Kinases/genetics , Protein Kinases/metabolism , Pyruvate Dehydrogenase Complex/genetics , Pyruvate Dehydrogenase Complex/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SpodopteraABSTRACT
The sequence motif commonly called a Nudix box, represented by (GX(5)EX(7)REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn(2+) and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K(m) value of 0. 36 mm. A V(max) of 12. 7 units mg (-1) for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.