Alternative Analytical Techniques for the Separation and Detection of Jasmonic Acid
Plants respond to several environmental stresses such as pathogen attack, mechanical or herbivorous insect-driven wounding and the presence of pollutants by the synthesis of secondary metabolites that mediate interplant communication for defense responses. Among these signalling molecules are phytohormones: jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). They are considered to be the major regulators of plant defense responses. The role of salicylic acid in influencing plant resistance to pathogens (aphids, bacteria, fungi) is well documented, whereas jasmonic acid is commonly believed to play an important role in plant responses to insect herbivores and abiotic stress. The soluble jasmonic acid acts as a direct defense to herbivores or others injuries, whereas the volatile form, methyl jasmonate, represents a plant signal molecule, which serves as an indirect defense mechanism.
The levels of jasmonic acid in plants vary with developmental stage, organs and species. Results obtained by several groups indicate that plants can have a jasmonic acid concentration between 10 and 1500 ng/g depending on the factors described above and physiological conditions.
Therefore, quantitative analysis of endogenous jasmonic acid is important for the physiological function of this compound and some different analytical techniques have been developed for assay of jasmonic acid.
Jasmonic acid has usually been detected by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) using specific antibodies. The main drawback of these methods is that all antibodies cross-react with other compounds of similar structure present in the same sample.
To overcome these shortcomings, several separation and detection techniques have been introduced for analytical assay of jasmonic acid:
Gas–liquid Chromatography – Mass Spectrometry (GC–MS)
High or Ultra-Performance Liquid Chromatography – Mass Spectrometry (HPLC–MS or UPLC-MS)
HPLC or UPLC with Fluorescence Detection. Derivatization of jasmonic acid from extract by fluorescence hydrazide
GC-FID with LC-MS as well to ID peaks in the extract is also a possibility; and finally
Although GC–MS and LC–MS enable reliable separation, identification and quantitation of compounds, complicated and time-consuming sample-cleaning steps, for example, the use of ionexchange columns or hydrophobic columns, are still needed before the derivatization step to ensure accurate quantitative analysis of jasmonic acid. It is, therefore, important to develop a simpler and faster analytical method for assay of small sample quantities.
Capillary Electrophoresis has been recognized as an important alternative method for the analysis of plant hormones as a result of its high performance, high speed and low sample consumption. Detection of attomole amounts is achieved when laser-induced fluorescence detection is introduced because jasmonic acid lacks a suitable fluorophore; however, detection of jasmonic acid by LIF requires derivatization.
It is possible to develop a Capillary Electrophoresis with Laser-Induced Fluorescence method for sensitive detection of jasmonic acid using a pre-column fluorescence labeling reagent 5-bromomethylfluorescein (5-BMF).
To find out more about how these techniques can be used, explore the case studies in: