QUANTIFICATION OF RAPID, TRANSIENT INCREASES IN JASMONIC ACID IN WOUNDED PLANTS USING A MONOCLONAL-ANTIBODY

A monoclonal antibody (MAB JAH1-8-B4) for the analysis of 3R, 7R-jasmonic acid and its methyl ester is described. An IgG1(kappa) immunoglobulin, MAB JAH1-8-B4, was used to set up a competitive enzyme-linked immunoassay employing 3R, 7R-jasmonate coupled to alkaline phosphatase as tracer. The assay has a linearity range (logit/log) between 50 fmol and 50 pmol (approx. 10 pg-10 ng) of 3R, 7R-methyljasmonate, the assay standard. A procedure combining prepurification of plant extracts by solid-phase extraction, followed by high-performance liquid chromatography and quantitation has been worked out, which uses 4 g of fresh plant material and has a detection limit between 0.2 and 0.4 mug of 3R, 7R-jasmonic acid (determined as its methyl ester) per kg of tissue, depending on the tissue. Internal standards of 3R, 7R-methyljasmonate, added to split samples during extraction as well as a second internal standard, 3R, 7R-methyljasmonate-[O-(CH3)-H-3], added to all samples prior to methylation, served to correct for workup losses and for the monitoring of chromatographic separations. Using this assay, it was found that levels of jasmonic acid rise immediately and transiently in the tissues analyzed as a consequence of wounding. These data provide further and direct evidence for the hypothesis that wound-induction of the plant defense reactions is mediated by endogenous jasmonates.