Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Abstract

Background: Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging.

Results: The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension.

Conclusions: This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Figure 1

Effects of solvents on hormone extraction. Concentration of endogenous plant hormones (A) Z, DHZ, 2iP, IAA, ACC, and (B) ABA, JA, SA, GA₄, GA₉, GA₂₄ detected in rosemary leaves using the following extraction solvents: M = methanol: glacial acetic acid, 99:1 (v/v); 80:20 M/I = methanol: isopropanol: glacial acetic acid 80:19:1 (v/v/v); 60:40 M/I = methanol: isopropanol: glacial acetic acid 60:39:1 (v/v/v); 40:60 M/I = methanol: isopropanol: glacial acetic acid 40:59:1 (v/v/v); 20:80 M/I = methanol: isopropanol: glacial acetic acid 20:79:1 (v/v/v); I = isopropanol: glacial acetic acid, 99:1 (v/v); 100:100 M/I = leaves were extracted first with methanol: glacial acetic acid, 99:1 (v/v) in two extraction steps and then with I = isopropanol: glacial acetic acid, 99:1 (v/v) in two extraction steps.

Figure 2

Effects of drying and re-suspension. Plant extract directly injected into the UPLC/ESI-MS/MS (F_INJ) compared to dried under nitrogen stream and re-suspended (D_INJ). Concentration of endogenous plant hormones (A) Z, DHZ, 2iP, IAA, ACC, and (B) ABA, JA, SA, GA₄, GA₉, GA₂₄ found in rosemary leaves.

Figure 3

Salt stress alters the hormonal balance of leaves. Concentration of endogenous plant hormones (A) Z, DHZ, 2iP, IAA, ACC, and (B) ABA, JA, SA, GA₄, GA₉, GA₂₄ found in rosemary leaves compared to control and salt-stressed plants (200 mM NaCl for 8 days). C.Y., young leaves of control plants; C.O., old leaves of control plants; S.Y., young leaves of salt-stressed plants; S.O., old leaves of salt-stressed plants.

Figure 4

Summary of the extraction protocol and analysis. Schematic extraction diagram to analyze small amounts of plant material using UPLC/ESI-MS/MS with multiple reaction monitoring (MRM).