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      Validated method for phytohormone quantification in plants

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          Abstract

          Phytohormones are long time known as important components of signaling cascades in plant development and plant responses to various abiotic and biotic challenges. Quantifications of phytohormone levels in plants are typically carried out using GC or LC-MS/MS systems, due to their high sensitivity, specificity, and the fact that not much sample preparation is needed. However, mass spectrometer-based analyses are often affected by the particular sample type (different matrices), extraction procedure, and experimental setups, i.e., the chromatographic separation system and/or mass spectrometer analyser (Triple-quadrupole, Iontrap, TOF, Orbitrap). For these reasons, a validated method is required in order to enable comparison of data that are generated in different laboratories, under different experimental set-ups, and in different matrices. So far, many phytohormone quantification studies were done using either QTRAP or Triple-quadrupole mass spectrometers. None of them was performed under the regime of a fully-validated method. Therefore, we developed and established such validated method for quantification of stress-related phytohormones such as jasmonates, abscisic acid, salicylic acid, IAA, in the model plant Arabidopsis thaliana and the fruit crop Citrus sinensis, using an Iontrap mass spectrometer. All parameters recommended by FDA (US Food and Drug Administration) or EMEA (European Medicines Evaluation Agency) for validation of analytical methods were evaluated: sensitivity, selectivity, repeatability and reproducibility (accuracy and precision).

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          Recent advances and emerging trends in plant hormone signalling.

          Plant growth and development is regulated by a structurally unrelated collection of small molecules called plant hormones. During the last 15 years the number of known plant hormones has grown from five to at least ten. Furthermore, many of the proteins involved in plant hormone signalling pathways have been identified, including receptors for many of the major hormones. Strikingly, the ubiquitin-proteasome pathway plays a central part in most hormone-signalling pathways. In addition, recent studies confirm that hormone signalling is integrated at several levels during plant growth and development.
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            Plant hormones are versatile chemical regulators of plant growth.

            The plant hormones are a structurally unrelated collection of small molecules derived from various essential metabolic pathways. These compounds are important regulators of plant growth and mediate responses to both biotic and abiotic stresses. During the last ten years there have been many exciting advances in our understanding of plant hormone biology, including new discoveries in the areas of hormone biosynthesis, transport, perception and response. Receptors for many of the major hormones have now been identified, providing new opportunities to study the chemical specificity of hormone signaling. These studies also reveal a surprisingly important role for the ubiquitin-proteasome pathway in hormone signaling. In addition, recent work confirms that hormone signaling interacts at multiple levels during plant growth and development. In the future, a major challenge will be to understand how the information conveyed by these simple compounds is integrated during plant growth.
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              Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

              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.
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                Author and article information

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                26 August 2014
                2014
                : 5
                : 417
                Affiliations
                [1] 1Department Bioorganic Chemistry, Max Planck Institute for Chemical Ecology Jena, Germany
                [2] 2LaBioMMi, Chemistry Department, Federal University of São Carlos São Carlos, Brazil
                [3] 3Accert Chemistry and Biotechnology Inc. São Carlos, Brazil
                Author notes

                Edited by: Erich Kombrink, Max Planck Institute for Plant Breeding Research, Germany

                Reviewed by: Stephan Pollmann, Universidad Politécnica de Madrid, Spain; Jonathan Michael Plett, University of Western Sydney, Australia

                *Correspondence: Axel Mithöfer, Department Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, Jena 07745, Germany e-mail: amithoefer@ 123456ice.mpg.de

                This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science.

                Article
                10.3389/fpls.2014.00417
                4143963
                25206356
                1943c221-7923-402d-bc19-b02c1aeea35c
                Copyright © 2014 Almeida Trapp, De Souza, Rodrigues-Filho, Boland and Mithöfer.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 30 April 2014
                : 05 August 2014
                Page count
                Figures: 4, Tables: 6, Equations: 0, References: 30, Pages: 11, Words: 8179
                Categories
                Plant Science
                Original Research Article

                Plant science & Botany
                phytohormones,hplc-ms/ms,quantification,arabidopsis thaliana,citrus sinensis,iontrap

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