methyl-jasmonate and capsidiol

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H(2)O(2) production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H(2)O(2) production is dependent on Ca(2+) influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca(2+) influx but independent of both H(2)O(2) production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H(2)O(2) and NO levels.

Topics: Acetates; Calcium; Cells, Cultured; Culture Media; Cyclodextrins; Cyclopentanes; Cytosol; Hydrogen Peroxide; Molecular Structure; Nicotiana; Nitric Oxide; Onium Compounds; Oxylipins; Phosphorylation; Plant Cells; Respiratory Burst; Sesquiterpenes; Signal Transduction

When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 microg/ml, the level of capsidiol was transiently increased in the culture media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor treatment doubled phospholiphase A(2) (PLA(2)) activity but simultaneous treatment with aristolochic acid, a PLA(2) inhibitor, inhibited sesquiterpenoid accumulation as well as PLA(2) activity. Mastoparan, a G protein activator, treatment also increased PLA(2) activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the C. annuum is mediated by PLA(2) activation.

Topics: Acetates; Aristolochic Acids; Capsicum; Cells, Cultured; Cellulase; Chromatography, Gas; Cyclopentanes; Intercellular Signaling Peptides and Proteins; Oxylipins; Peptides; Phospholipase A2 Inhibitors; Phospholipases A2; Sesquiterpenes; Time Factors; Wasp Venoms

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Topics: Acetates; Amino Acid Sequence; Base Sequence; Carbon-Carbon Lyases; Cells, Cultured; Cellulase; Cyclopentanes; DNA Primers; DNA, Plant; Fungal Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Molecular Sequence Data; Nicotiana; Oxylipins; Plant Growth Regulators; Plants, Toxic; RNA, Messenger; RNA, Plant; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sesquiterpenes

The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the beta-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

Topics: Acetates; Algal Proteins; Base Sequence; Carbon-Carbon Lyases; Cyclopentanes; DNA Mutational Analysis; Enzyme Induction; Fungal Proteins; Gene Expression Regulation, Plant; Genes, Reporter; Molecular Sequence Data; Nicotiana; Oxylipins; Plant Diseases; Plant Growth Regulators; Plants, Toxic; Promoter Regions, Genetic; Pseudomonas; Salicylates; Salicylic Acid; Sequence Deletion; Sesquiterpenes; Tissue Distribution; Transcription, Genetic; Transformation, Genetic