Monitoring Plant Hormones During Stress Responses

Summary

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Abstract

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

Protocol

1. First we need to prepare the 2ml screw-cap vials for extraction: Add 400ul extraction buffer (1-propanol:H2O:concentrated HCl 2:1:0.002 vol/vol/vol) and 10 μl of the respective internal standard (10ng/μl) to a vial. Freeze in liquid nitrogen and then add the ceramic beads.
2. Add the plant material (between 50 and 200mg) while the vials are still positioned in the liquid nitrogen. Note that the weight of the added plant material has to be estimated before further processing to allow later for exact quantification, which is based on the fresh weight.
3. Make sure that all liquid nitrogen is evaporated before tightly closing the vial with a cap. Note that the cap needs to....

Discussion

The method presented here has been proven to work reliably for a wide array of plant signaling compounds provided that the size and the chemical nature of the compound does not prevent it from being extracted, methylated, and vaporized. Also, the methylation procedure as it is described herein is not the only way of derivatization. Silyllation and acetylation are alternative methods and protocols can be easily found online.

If the mass spectrometer is not capable of single ion monitoring the m.......