okadaic-acid and jasmonic-acid

We report isolation and transcriptional profiling of rice (Oryza sativa L.) mitogen-activated protein kinase (MAPK), OsSIPK (salicylic acid-induced protein kinase). OsSIPK gene is located on chromosome 6 most probably existing as a single copy in the rice genome, and encodes 398 amino acid polypeptide having the MAPK family signature and phosphorylation activation motif TEY. Steady state mRNA analyses of OsSIPK showed weak constitutive expression in leaves of 2-week-old rice seedlings. A time course (30-120 min) experiment using a variety of elicitors and stresses revealed that the OsSIPK mRNA is strongly induced by jasmonic acid (JA), salicylic acid (SA), ethephon, abscisic acid, cycloheximide (CHX), JA/SA + CHX, cantharidin, okadaic acid, hydrogen peroxide, chitosan, sodium chloride, and cold stress (12 degrees C), but not with wounding by cut, gaseous pollutants ozone, and sulfur dioxide, high temperature, ultraviolet C irradiation, sucrose, and drought. Its transcription was also found to be tissue-specifically regulated, and followed a rhythmic dark induction in leaves. Finally, we showed that the OsSIPK protein is localized to the nucleus. From these results, OsSIPK can be implicated in diverse stimuli-responsive signaling cascades and transcription of certain genes.

Topics: Abscisic Acid; Blotting, Northern; Cantharidin; Cell Nucleus; Chitosan; Cycloheximide; Cyclopentanes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hydrogen Peroxide; Mitogen-Activated Protein Kinases; Okadaic Acid; Organophosphorus Compounds; Oryza; Oxylipins; Plant Leaves; Plant Proteins; RNA, Messenger; Salicylic Acid; Seedlings; Sequence Analysis, DNA; Sulfur Dioxide; Ultraviolet Rays

Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC) and Escherichia coli beta-glucuronidase (GUS) reporters were expressed under control of the jasmonate-responsive LOX2 promoter. Spatial and temporal patterns of reporter expression were determined initially, and revealed that JA-responsive expression from the LOX2 promoter required de novo protein synthesis. Reporter activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid. FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition, reporter induction and endogenous LOX2 expression by staurosporine was absent in joe2.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclopentanes; Escherichia coli; Gene Expression Regulation; Glucuronidase; Lipoxygenase; Luciferases; Luminescent Measurements; Mutation; Okadaic Acid; Oxylipins; Phenotype; Phosphoric Monoester Hydrolases; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified; Promoter Regions, Genetic; Protein Kinase Inhibitors; Protein Kinases; Recombinant Fusion Proteins; RNA, Messenger; Signal Transduction; Staurosporine

A novel rice (Oryza sativa L.) gene, homologous to a sorghum pathogenesis-related class 10 protein gene, was cloned from a cDNA library prepared from 2-week-old jasmonic acid-treated rice seedling leaves, and named as JIOsPR10 (jasmonate inducible). JIOsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17,173.23 Da and a pI of 5.84. JIOsPR10 was highly similar (77%) to the sorghum PR10 protein, but showed less than 55% similarity with other identified PR10s at the amino acid level. Genomic Southern analyses indicated the presence of related genes in the rice genome. The JIOsPR10 transcript was not detected in the healthy leaves, and was not induced after cut. Further expression analysis revealed that the signaling components of defense/stress pathways, jasmonate, salicylate, and H(2)O(2) significantly up-regulated the JIOsPR10 mRNA over the cut control, whereas two other stress regulators, ethylene and abscisic acid, failed to induce its expression. Interestingly the protein phosphatase (PP) inhibitors, cantharidin, endothall, and okadaic acid, rapidly and potently up-regulated the JIOsPR10 expression, suggesting involvement of the phosphorylation/dephosphorylation events. Additionally, the inducible expression of the JIOsPR10 gene was influenced by light signal(s). Finally, the blast pathogen (Magnaporthe grisea) also specifically elicited the accumulation of JIOsPR10 mRNA in leaves. Induction of the JIOsPR10 gene expression by signaling molecules, PP inhibitors and pathogen attack, strongly indicate a role for this novel gene in rice self-defense/stress response(s).

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Southern; Cantharidin; Cloning, Molecular; Cyclopentanes; Dicarboxylic Acids; DNA, Complementary; Enzyme Inhibitors; Gene Library; Genome, Plant; Hydrogen Peroxide; Molecular Sequence Data; Okadaic Acid; Oryza; Oxylipins; Peptides; Phosphoprotein Phosphatases; Phosphorylation; Phylogeny; Plant Leaves; Plant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salicylic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Signal Transduction; Time Factors; Up-Regulation

Plants responses to mechanical injury are complex and include the induced expression of defence-related genes. The phytohormone JA has been reported to mediate some of these responses. To elucidate further the signal transduction processes involved, the action of specific agonists and antagonists of known signalling effectors on the response of Arabidopsis thaliana plantlets to JA and wounding was investigated. The identification and characterization of a reversible protein phosphorylation step in a transduction pathway leading to JA-induced gene transcription is reported. This phosphorylation event involved the opposing activities of a staurosporine-sensitive protein kinase, negatively regulating the pathway, and a protein phosphatase, most probably of type 2 A, which activated JA-responsive gene expression. JA activation via this pathway was blocked in the A. thaliana JA-insensitive mutants jin1, jin4 and coi1, and by exogenous application of cycloheximide or auxins. Wound-induced activation of JA-responsive genes was also regulated by this protein phosphorylation step. An alternative wound signalling pathway, independent of JA, was also identified, leading to the transcriptional activation of a different set of genes. This JA-independent pathway was also regulated by a protein phosphorylation switch, in which the protein kinase positively regulated the pathway while the protein phosphatase negatively regulated it. Moreover, a labile protein apparently repressed the expression of these genes. One of the genes analysed, JR3, had a complex pattern of expression, possibly because it was regulated via both of the wound signalling pathways identified. According to the function of an homologous gene, JR3 may be involved in feedback inhibition of the JA response.

Topics: Arabidopsis; Cycloheximide; Cyclopentanes; Enzyme Inhibitors; Feedback; Gene Expression Regulation, Plant; Genes, Plant; Indoleacetic Acids; Models, Biological; Okadaic Acid; Oxylipins; Phosphoprotein Phosphatases; Phosphorylation; Plant Growth Regulators; Plant Proteins; Protein Kinase Inhibitors; Protein Kinases; Signal Transduction; Staurosporine; Transcriptional Activation