|Tumlinson Iii, James|
Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2003
Publication Date: 9/2/2003
Citation: Schmelz, E.A., Engelberth, J.E., Alborn, H.T., O'Donnell, P., Sammons, M., Toshima, H., Tumlinson III, J.H. 2003. Simultaneous analysis of phytohormones, phytotoxins and volatile organic compounds in plants. Proceedings of the National Academy of Sciences. 100(18):10552-10557 Interpretive Summary: Highly specific plant defense responses to biotic stress, such as insect and pathogen attack, are regulated through the coordinated interaction of phytohormone signals, referred to as signaling crosstalk. A significant obstacle in understanding how multiple phytohormones control specific plant responses has been the quantification of these interacting signals. 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, 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 are converted into volatile methyl esters and easily separated from the extraction mixture by collecting the analytes as volatiles. Samples are separated using gas chromatography and quantified using chemical ionization mass spectrometry. The versatility of the method is further demonstrated by the additional analysis of pathogen derived elicitors and insect-induced volatile organic compounds (VOC). Pathogen elicitors, such as coronatine from Pseudomonas syringe, can mimic phytohormones and mediate the progression of disease symptoms. Likewise, in response to caterpillar attack, plants emit VOC that aid in the attraction of natural enemies of the insect pests. This facile and rapid method for analyzing a wide range of phytohormones, elicitors, and chemical signals will enable elucidation of key crosstalk interactions and identify molecular targets for increasing the inducible expression of plant defenses.
Technical Abstract: Phytohormones regulate the protective responses of plants against both biotic and abiotic stresses through synergistic or antagonistic actions referred to as signaling crosstalk. A bottleneck in crosstalk research is the quantification of numerous interacting phytohormones and regulators. The chemical analysis of salicylic acid (SA), jasmonic acid (JA), indole-3-acetic acid (IAA), and abscisic acid (ABA) is typically achieved using separate and complex methodologies. Moreover, pathogen produced phytohormone mimics, such as the phytotoxin coronatine (COR), have not been directly quantified in plant tissues. We address these problems using a simple preparation and a GC-MS based metabolic profiling approach. Plant tissue is extracted in aqueous propanol and mixed with dichloromethane. Carboxylic acids present in the organic layer are methylated using trimethylsilyl diazomethane, analytes are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a sample vial. Analytes are separated using gas chromatography and quantified using chemical ionization mass spectrometry that produces predominantly [M+H]+ parent ions. We use this technique to examine levels of COR, phytohormones, and volatile organic compounds (VOC) in model systems including Arabidopsis thaliana during infection with Pseudomonas syringe pv. tomato DC3000, corn (Zea mays) under herbivory by corn earworm (Helicoverpa zea), tobacco (Nicotiana tabacum) following mechanical damage and tomato (Lycopersicon esculentum) during drought stress. Numerous complex changes induced by pathogen infection including the accumulation of COR, SA, JA, IAA, and ABA and illustrate the potential and simplicity of this approach in quantifying signaling crosstalk interactions that occur at the level of synthesis and accumulation.