Open-i Logo
Submit this form
Results 1-1   << Back

 
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).
© Copyright Policy

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).

Mentions: The development of a rapid, sensitive, high throughput, cost effective method for quantification of 17 endogenous plant hormones from seven plant classes in a complex matrix is described. The use of UPLC/ESI-MS/MS with multiple reaction monitoring (MRM) allowed each sample could be analyzed within 6 minutes. The method requires minimal plant tissue, is highly reproducible and was applicable to analyze dynamic changes in endogenous concentrations of hormones in plants exposed to salt stress. Due to the structural diversity of plant hormones no one extraction solvent was capable of extract all plant hormones equally well. If we aim at reducing extraction steps to a minimum, depending on the plant hormones of interest a different solvent is recommended: methanol:isopropanol:glacial acetic acid, 20:79:1 (v/v/v) for ABA, SA, JA, IAA and GAs, methanol:isopropanol:glacial acetic acid, 60:39:1 (v/v/v) for cytokinins, and methanol:glacial acetic acid, 99:1 (v/v) for ACC. Furthermore, this method is equally applicable to fresh or freeze dried tissue. Moreover, high loss of plant hormones during drying of sample extracts under N2 and re-suspension of the residues indicates that sample extracts should immediately be injected. However, the adjustment of sample weight and volume of the extraction solvent must be taken into account. As a summary, a schematic extraction diagram to analyze small amounts of plant material without time-consuming additional steps such as purification, sample drying under N2 and re-suspension of the residues using UPLC/ESI-MS/MS with MRM is shown in Figure 4. This method has been used in our group to obtain the hormone profile of a number of plant tissues, including leaves, flowers and seeds of different species of a number of plant families (including the model plant Arabidopsis thaliana and other Brassicaceae, Liliaceae, Cistaceae, Anacardiaceae, Dioscoreaceae, Velloziaceae, Xyridaceae and Lamiaceae) that all bring completely different matrices and therefore their specific complexity for analyses. As shown in the present study, each species will require a specific handling during extraction and analysis, and the schematic diagram presented in Figure 4 can serve as a basis to the optimization of the method for each species. Although obtaining the hormone profile of species with complex matrices may be more difficult due to the presence of possible interfering compounds, it is also challenging and will undoubtedly be needed in the near future if we are to better understand plant stress responses in species of contrasting habitats that can be used as model plants for the study of plant responses to environmental stresses.

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

Müller M, Munné-Bosch S - Plant Methods (2011)

Bottom Line: 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.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.

Affiliation: Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, E-08028 Barcelona, Spain. smunne@ub.edu.

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.

View Similar Images In: Results Collection              View Article: Pubmed Central HTML PubMed      Show All Figures 
getmorefigures.php?pmc=3253682&rFormat=json&query=null&fields=all&favor=none&it=none&sub=none&sp=none&coll=none&req=5
Show MeSH

Lister Hill National Center for Biomedical Communications
U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894
National Institutes of Health, Department of Health & Human Services
Privacy, Accessibility, Frequently Asked Questions, Contact Us, Collection
Freedom of Information Act, USA.gov