Poster: Emerging Technologies

Abs # 873: Simultaneous analysis of phytohormones, pathogen elicitors and volatile secondary metabolites from plants using vapor phase extraction and chemical ionization GC-MS

Presenter:	Schmelz, Eric A, eschmelz@gainesville.usda.ufl.edu
Authors	Schmelz, Eric A (A)   Engelberth, Juergen  (A)   Alborn, Hans T (A)   Tumlinson, James H (A)
Affiliations:	(A): USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology

Hormones regulate growth, reproduction and the protective responses of plants against biotic and abiotic stresses. How hormones interact, either synergistically or antagonistically, to coordinate these responses is referred to as signaling crosstalk. A current bottleneck in crosstalk research is the difficulty in quantifying numerous potentially interacting phytohormone signals and pathogen elicitors. Many methods used to quantify diverse regulators such as salicylic acid (SA), jasmonic acid (JA), indole-3-acetic acid (IAA), abscisic acid (ABA), and coronatine use separate, tedious and complex purification schemes. We address this problem using a novel preparation scheme and a GC-MS based metabolic profiling approach. Plant tissue is extracted in acidified aqueous propanol and mixed with dichloromethane. Carboxylic acids present in the organic layer are methylated using trimethylsilyldiazomethane, low molecular weight components are volatilized under heat, collected on a polymeric absorbent, and eluted with solvent into a GC/MS vial. The procedure produces a complex chromatogram that is separable through the use of chemical ionization that produces predominantly M+1 parent ions. We demonstrate the use of this high-recovery technique by examining changes in benzoic acid (BA), SA, JA, IAA, ABA, coronatine and other secondary metabolites in model systems including tomato (Lycopersicon esculentum) during draught stress, tobacco (Nicotiana tabacum) following mechanical damage, corn (Zea mays) under herbivory by corn earworm (CEW, Helicoverpa zea), and Arabidopsis thaliana during pathogen infection with Pseudomonas syringae. Numerous changes induced by Pseudomonas infection including SA, JA, IAA, ABA and coronatine illustrate the potential and simplicity of this approach.