alamethicin and jasmonic-acid

Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175μM and 341μM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

Topics: Acetates; Alamethicin; Benzoic Acid; Biocatalysis; Catalytic Domain; Cyclopentanes; Escherichia coli; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hydrogen-Ion Concentration; Kinetics; Metals; Methyltransferases; Multigene Family; Mutation; Oxylipins; Phylogeny; Plant Growth Regulators; Plant Proteins; Populus; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Salicylic Acid; Substrate Specificity; Temperature

The induction of plant defences involves a sequence of steps along a signal transduction pathway, varying in time course. In this study, the effects of induction of an early and a later step in plant defence signal transduction on plant volatile emission and parasitoid attraction are compared. Ion channel-forming peptides represent a class of inducers that induce an early step in signal transduction. Alamethicin (ALA) is an ion channel-forming peptide mixture from the fungus Trichoderma viride that can induce volatile emission and increase endogenous levels of jasmonic acid (JA) and salicylic acid in plants. ALA was used to induce an early step in the defence response in Brussels sprouts plants, Brassica oleracea var. gemmifera, and to study the effect on volatile emission and on the behavioural response of parasitoids to volatile emission. The parasitoid Cotesia glomerata was attracted to ALA-treated plants in a dose-dependent manner. JA, produced through the octadecanoid pathway, activates a later step in induced plant defence signal transduction, and JA also induces volatiles that are attractive to parasitoids. Treatment with ALA and JA resulted in distinct volatile blends, and both blends differed from the volatile blends emitted by control plants. Even though JA treatment of Brussels sprouts plants resulted in higher levels of volatile emission, ALA-treated plants were as attractive to C. glomerata as JA-treated plants. This demonstrates that on a molar basis, ALA is a 20 times more potent inducer of indirect plant defence than JA, although this hormone has more commonly been used as a chemical inducer of plant defence.

Topics: Alamethicin; Animals; Brassicaceae; Cyclopentanes; Eating; Hymenoptera; Oxylipins; Signal Transduction; Species Specificity; Volatilization

Jasmonic acid and salicylic acid represent important signaling compounds in plant defensive responses against other organisms. Here, we present a new method for the easy, sensitive, and reproducible quantification of both compounds by vapor-phase extraction and gas chromatography-positive ion chemical ionization-mass spectrometry. The method is based on a one-step extraction, phase partitioning, methylation with HCl/methanol, and collection of methylated and, thus, volatilized compounds on Super Q filters, thereby omitting further purification steps. Eluted samples are analyzed and quantified by GC/MS with chemical ionization. Standard curves were linear over a range of 5-1000 ng for jasmonic acid and salicylic acid. The correlation coefficients were greater than 0.999 and the recovery rates estimated between 70 and 90% for salicylic acid and 90 and 100% for jasmonic acid. The limit of detection was about 500 fg by using single ion detection mode. Both, cis- and trans-isomers for jasmonic acid can be detected. A comparison with established methods indicates the new method to be highly efficient, allowing reliable quantification of both compounds from small amounts of plant material (5-400mg fresh weight).

Topics: Alamethicin; Animals; Arachis; Cyclopentanes; Gas Chromatography-Mass Spectrometry; Gases; Nicotiana; Oxylipins; Plant Extracts; Plants; Salicylic Acid; Sensitivity and Specificity; Spodoptera; Zea mays

The perception of thigmic stimuli is a widespread phenomenon among plants with decisive meaning for the ability to survive. Beside a general sensitivity for mechanical stimuli many plants have evolved specialized organs with highly developed mechanisms to perceive and transduce the applied forces. Tendrils of Bryonia dioica and Pisum sativum have been chosen to study the effects of mechanical stimulation on plant physiology. Both types of tendrils, although exhibiting different morphology, respond to such a stimulus with a rapid coiling response to the dorsal side of the organ within minutes. The actual perception of the stimulus is most likely coupled to the cytoskeleton serving as the mediator between the physical stimulus and the biochemical response. Drugs affecting the status of the cytoskeleton were used to get more insights into this specific process. The results indicate that microtubuli (MT) play the most important role in the perception of thigmic stimuli in tendrils. Colchicine-mediated disruption of MT lead to total inhibition of the response to the thigmic stimulus in tendrils of Pisum and to a reduced response in Bryonia. Alamethicin, an ionophore that can mimic action potentials in membranes, was able to bypass this inhibition suggesting a direct involvement of MT in depolarization of the membranes. Auxin, however, which is also supposed to be involved in the regulation of the coiling response, failed to bypass colchicine-dependent inhibition. Vinblastine, another microtubule depolimerizing agent, did induce tendril coiling in Pisum without further stimulation. Application of taxol and other MT-stabilizing drugs as well as disruption of the actin network did not affect the coiling response of tendrils. In Pisum indole-3-acetic acid (IAA) is induced after mechanical stimulation during the coiling response, but not jasmonic acid. A further consequence of mechanical stimulation is the induction of an oxidative burst and an increase in soluble sugar. A model is presented integrating these results and might serve as a common basis for the understanding of the perception of mechanical stimuli.

Topics: Alamethicin; Bryonia; Cell Wall; Colchicine; Cyclopentanes; Cytochalasin D; Cytoskeleton; Indoleacetic Acids; Mechanotransduction, Cellular; Microtubules; NAD; NADPH Oxidases; Oxylipins; Paclitaxel; Pisum sativum; Plant Components, Aerial; Plant Growth Regulators; Signal Transduction; Stress, Mechanical; Tubulin Modulators; Uncoupling Agents; Vinblastine

Alamethicin (ALA), a voltage-gated, ion channel-forming peptide mixture from Trichoderma viride, is a potent elicitor of the biosynthesis of volatile compounds in lima bean (Phaseolus lunatus). Unlike elicitation with jasmonic acid or herbivore damage, the blend of substances emitted comprises only the two homoterpenes, 4,11-dimethylnona-1,3,7-triene and 4,8,12-trimethyltrideca-1,3,7,11-tetraene, and methyl salicylate. Inhibition of octadecanoid signaling by aristolochic acid and phenidone as well as mass spectrometric analysis of endogenous jasmonate demonstrate that ALA induces the biosynthesis of volatile compounds principally via the octadecanoid-signaling pathway (20-fold increase of jasmonic acid). ALA also up-regulates salicylate biosynthesis, and the time course of the production of endogenous salicylate correlates well with the appearance of the methyl ester in the gas phase. The massive up-regulation of the SA-pathway (90-fold) interferes with steps in the biosynthetic pathway downstream of 12-oxophytodienoic acid and thereby reduces the pattern of emitted volatiles to compounds previously shown to be induced by early octadecanoids. ALA also induces tendril coiling in various species like Pisum, Lathyrus, and Bryonia, but the response appears to be independent from octadecanoid biosynthesis, because inhibitors of lipoxygenase and phospholipase A(2) do not prevent the coiling reaction.

Topics: Alamethicin; Cyclopentanes; Fatty Acids, Volatile; Ion Channels; Kinetics; Oxylipins; Phaseolus; Plant Growth Regulators; Plant Leaves; Salicylates; Signal Transduction