Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2004
Publication Date: 9/20/2004
Citation: Schmelz, E.A., Engelberth, J., Tumlinson, J.H., Block, A., Alborn, H.T. 2004. The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolities. Plant Journal. 39(5):790-808. Interpretive Summary: Rapid plant responses that serve as protective mechanisms against both biotic and abiotic stresses, such as insect/pathogen attack and drought, are regulated through the coordinated interaction of phytohormone signals and subsequent changes in primary and secondary metabolites. The emerging field of metabolomics seeks to quantify changes in all plant metabolites in effort to understand this dynamic physiology and function. A significant obstacle in the expansion of metabolic profiling analyses has been the simultaneous quantification of both low abundance phytohormones as well as predominant primary and secondary metabolites. Scientists at the Center for Medical, Agricultural and Veterinary Entomology in Gainesville, FL, have discovered a simple and rapid method for the preparation of samples and simultaneous analysis of multiple plant hormones, fatty acids, pathogen derived elicitors, and volatile organic compounds present in plants. Relatively non-volatile acidic phytohormones such as salicylic acid, jasmonic acid, indole-3-acetic acid, abscisic acid and 12-oxo-phytodienoic acid are converted into volatile methyl esters and separated from the complex extraction mixture by collecting the compounds as volatiles. Samples are separated using gas chromatography and quantified using chemical ionization mass spectrometry. We now provide a detailed description of this facile and rapid method for analyzing a phytohormones, elicitors, chemical signals, and a wide range of metabolites. Such approaches will enable the elucidation of key phytohormone interactions and identify molecular targets for increasing the expression of plant defenses.
Technical Abstract: Through complex networks of signaling interactions, phytohormones regulate growth, development, reproduction and responses to biotic and abiotic stress. Comprehensive metabolomic approaches, seeking to quantify changes in vast numbers of plant metabolites, may ultimately clarify these complex signaling interactions and consequently explain pleiotropic effects on plant metabolism. Synergistic and antagonistic phytohormone signaling interactions, referred to as crosstalk, are often considered at the level of transduction without proper consideration of synthesis or accumulation of phytohormones due to the limitation and difficulty in quantifying numerous signals. Significant progress has recently been made in the expansion of metabolic profiling and analysis of multiple phytohormones (Müller et al., 2002, Birkemeyer et al., 2003., Chiwocha et al., 2003, Schmelz et al., 2003). We recently presented a novel metabolic profiling approach to the analysis of acidic phytohormones and other metabolites based on a simplistic preparation scheme and analysis by chemical ionization-gas chromatography/mass spectrometry (CI-GC/MS). We now provide a detailed description of this vapor phase extraction (VPE) technique and use pathogen infection of Arabidopsis with Pseudomonas syringae DC3000 to illustrate metabolic changes in salicylic acid (SA), cinnamic acid (CA), jasmonic acid (JA), indole-3-acetic acid (IAA), abscisic acid (ABA), unsaturated C18 fatty acids (FA), 12-oxo-phytodienoic acid (OPDA), and phytotoxin coronatine (COR). Directions for further method expansion are provided and include issues of recovery, derivatization, range of accessible analytes, optimization, reproducibility and future directions.