Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry
© Müller and Munné-Bosch; licensee BioMed Central Ltd. 2011
Received: 12 July 2011
Accepted: 18 November 2011
Published: 18 November 2011
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.
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.
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.
KeywordsUPLC/ESI-MS/MS Phytohormones Auxins Abscisic acid Cytokinins Gibberellins Salicylic acid Jasmonic acid 1-amino-cyclopropane-1-carboxyic acid Rosmarinus officinalis
collision energy: CXP: collision cell exit potential
electrospray ionization tandem mass spectrometry
isopentenyladenine: IPA: isopentenyladenosine
limit of detection
limit of quantification
multiple reaction monitoring
relative deviation standard
ultrahigh performance liquid chromatography
Hormones play a pivotal role in most physiological processes in plants. These structurally diverse compounds that act usually at nanomolar levels include five groups of the so-called "classic" hormones, comprising auxins, cytokinins, gibberellins (GA), abscisic acid (ABA) and ethylene, and several other plant growth regulators, including jasmonates, salicylates, brassinosteroids, polyamines or the very recently discovered strigolactones, which fit several of the criteria to be considered hormones [1–3]. Furthermore, the list of plant hormones is expected to increase due to a better understanding of plant growth and development and stress responses, and the use of technological advances in analytical methods.
Recent studies support the contention that hormone actions build a signaling network and mutually regulate several signaling and metabolic systems, such as auxins and GAs in growth regulation , CKs, auxins, ABA and strigolactones in apical dominance [2, 5], auxins and brassinosteroids in cell expansion [6, 7], ethylene and cytokinins in the inhibition of root and hypocotyl elongation , ethylene, ABA and GAs in some plant stress responses [9, 10], or SA, JA and auxin in plant responses to pathogens [11, 12] to name just a few of the reported hormonal interactions. Therefore, focusing on a single endogenous plant hormone to evaluate hormone-regulated physiological or developmental biological problems is not sufficient anymore .
In order to understand better the network regulation of hormone action influencing plant growth and development as well as the distribution of several hormones at the organ, cellular and sub-cellular levels, an ideal analytical method should provide a measure of multiple hormone concentrations (hormonal profiling) from a single experimental sample. Therefore several methods for the simultaneous quantification of multiple plant hormones using mass spectrometry with multiple reaction monitoring (MRM) have been developed recently. It has been reported a multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique for the simultaneous analysis of SA, JA, IAA, ABA and OPDA in Arabidopsis thaliana. However, GC-MS is limited to volatile compounds and as a result it is necessary to purify and derivatize hormones prior to analysis. Another potential downside in GC-MS procedures apart from the purification and derivatization is the use of high temperatures, which can degrade thermal labile compounds .
An alternative to GC-MS is liquid chromatography coupled to mass spectrometry (LC-MS). A high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous analysis of 15 plant hormones and metabolites from four different hormone classes (auxins, cytokinins, GAs and ABA) has been reported to analyze hormone regulation of thermodormancy of lettuce seeds . Also, a HPLC/ESI-MS/MS method to analyze seven major classes of plant hormones including auxins, cytokinins, GAs, ABA, jasmonates, brassinosteriods and SA in Arabidopsis thaliana has been developed . Furthermore, an ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) technique to analyze cytokinins, auxins, ABA and GAs in rice has been described . To improve the detection limit of the negatively charged compounds they derivatized auxin, ABA and GAs with bromocholin and analyzed all compounds in the positive ion mode. However, at present this method is limited and cannot target other plant hormones such as JA and SA.
Plant hormones are structurally diverse compounds with diverse physiochemical properties. The question as to whether all plant hormones can be extracted equally well has not yet been answered. The choice of extraction methods depends not only on the target analysts but also on the matrix of the analyzed tissues. The requirements on the extraction method increase with the complexity of the sample matrix. In the literature diverse extraction solvents such as methanol, methanol-water mixtures, isopropanol, or isopropanol-water mixtures have been used with one or two extraction steps [14, 15, 17–19]. In addition, time-consuming multiple steps of sample preparation procedures, including sample purification, drying of sample under N2 and re-suspension of the residues have been reported for plant hormone extraction [14, 20] which may increase the risk of hormone loss. However, the application of internal standards can provide corrections for hormone loss during sample preparation and chromatographic separation.
Here we developed a new method which allows to analyze dynamic changes in endogenous concentrations of major plant hormones and to study plant development processes or plant responses to biotic and abiotic stresses in complex sample matrices. An example is shown in which rosemary (Rosmarinus officinalis), an aromatic Mediterranean perennial shrub rich in secondary metabolites and epicuticular waxes, was exposed to salt stress. Soil salinity is one of the most serious environmental threats for plant survival and affects many undesirable changes in plants such as hyperionic and hyperosmotic effects, increase in reactive oxygen species and metabolic toxicity. These changes lead to growth reduction, changes in biomass allocation and phenology, leaf senescence, and finally to plant death [21–23]. It has been shown that senescence induced by salinity follows at least in part similar physiological events as drought-induced senescence . Plant hormones such as ABA, ethylene and cytokinins are involved in different plant strategies to overcome the damaging effects of salinity, however, the complex hormonal response is only partly known [25, 26]. The present work reports a sensitive and rapid method to quantify 17 plant hormones from seven plant classes including auxins, cytokinins, GAs, ABA, ACC (the ethylene precursor), SA and JA in complex tissues using ultra-performance liquid chromatography mass spectrometry (UPLC/ESI-MS/MS) with multiple reaction monitoring (MRM). This method allows obtaining a hormonal profiling in 6 min. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized.
Results and discussion
Of the 17 endogenous plant hormones investigated, Z, DHZ, 2-IP, IAA, ABA, JA, SA, ACC, GA4, GA9, GA24 were detected in rosemary leaves, whereas ZR, DHZR, IPA, GA1, GA19, and GA20 concentrations were under the limit of detection. However, the internal standards d4-SA, d6-ABA, d5-JA, d5-IAA, d2-GA1, d2-GA4, d2-GA9, d2-GA19, d2-GA20, d2-GA24, d4-ACC, d6-2iP, d6-IPA, d5-Z and d5-ZR were detected in all rosemary leaf extracts (d5-Z and d5-ZR were used as internal standards for Z, DHZ and ZR, DHZR).
The extraction of plant hormones will critically determine the quality of the results obtained. Therefore the choice of the extraction solvent is very important, however, it is also challenging by the structurally diversity of plant hormones. Previously reported methods for plant hormone extraction used predominately methanol, methanol mixtures or isopropanol. Four classes of plant hormones including auxins, cytokinins, ABA and gibberellins were extracted using isopropanol:glacial acetic acid (99:1; v/v) . Methanol:water:acetic acid (10:79:1) was used to extract ABA, SA and JA . In other studies methanol:water:formic acid (75:20.5) was used to extract cytokinins, IAA and ABA [17, 19]. We tested methanol:glacial acetic acid, 99:1 (v/v), isopropanol:glacial acetic acid, 99:1 (v/v) and different methanol:isopropanol:glacial acetic acid mixtures, 80:19:1; 60:39:1; 40:59:1; 20:79:1 (v/v/v). Thirty-five 100 mg samples of frozen rosemary leaves were extracted with 7 different extraction solvents including 5 replicates after incorporation of deuterated labeled plant hormones as internal standards.
Fresh or dried leaf material
Little is known whether freeze drying (compared to fresh plant material) adversely affects plant hormone contents. A 25% decrease was observed for SA and JA yields from freeze dried compared to fresh leaf material of Arabidopsis. A decrease of 50% in SA but no change in JA levels of freeze dried material from cucumber compared to those from the equivalent amount of fresh tissue was measured . Here plant hormone contents from fresh frozen and freeze dried leaf material of rosemary were compared. Leaves were collected and immediately frozen in liquid nitrogen. Ten fresh weight (FW) and ten freeze dried samples (DW) were then extracted after the addition of internal standards. Additional File 1 shows no significant differences in plant hormone contents comparing fresh frozen and freeze dried plant material, except for GA9, which showed significant higher contents in fresh samples.
Undoubtedly, the requirements on the extraction methods increase with the complexity of the sample matrix. Rosemary leaves represent a complex matrix including essential oils, tannins, flavonoids, diterpenes, saponins, epicuticular waxes and resin. Five 100 mg samples (fresh weight) were extracted five times after including internal standards. Each supernatant was immediately dried under nitrogen stream, re-suspended and injected to LC-MS. Additional File 2 shows clear differences regarding the necessary extraction steps for endogenous plant hormones. DHZ was only detectable in the first three extractions; 2iP, JA, and GA9 in four extractions; and Z, IAA, ACC, ABA, SA, GA4 and GA24 in five extractions.
In the optimum LC-MS/MS conditions calibration curves were created using solutions containing varying amounts of each unlabeled analyte compound and a known fixed amount of deuterium labeled internal standard. The obtained calibration curves showed linearity of correlation coefficients (R2) in the concentration range selected between 0.996 and 0.999 for the different analysts.
Reproducibility of the developed LC/ESI-MS/MS method.
Z/d 5 -Z
IAA/d 5 -IAA
ABA/d 6 -ABA
JA/d 5 -JA
SA/d 4 -SA
ACC/d 4 -ACC
GA 4 /d 2 -GA 4
GA 9 /d 2 -GA 9
GA 24 /d 2 -GA 24
LOD and LOQ values.
Requirements of plant material for the UPLC-MS/MS analysis of endogenous plant hormones in Rosmarinus officinalis plants.
Minimum tissue requirement
Plant hormones remain stable 48 h after extraction.
Z/d 5 -Z
IAA/d 5 -IAA
ABA/d 6 -ABA
JA/d 5 -JA
SA/d 4 -SA
ACC/d 4 -ACC
GA 4 /d 2 -GA 4
GA 9 /d 2 -GA 9
GA 24 /d 2 -GA 24
Hormonal profiling of rosemary leaves under salt stress
Relative water content (RWC) and maximum efficiency of PSII photochemistry (Fv/Fm ratio, indicative of damage to PSII) in Rosmarinus officinalis leaves of control and salt-stressed plants (treated with 200 mM NaCl for 8 days).
Control young leaves
80.83 ± 1.63a
0.837 ± 0.02a
Control old leaves
83.09 ± 1.57a
0.837 ± 0.01a
Stress young leaves
73.83 ± 1.78b
0.728 ± 0.03b
Stress old leaves
61.32 ± 1.10c
0.633 ± 0.04b
Special mention deserves the case of GAs, since more than 100 GAs are found in plants, although only a few of these are known to have biological activity such as GA4. Of the six analyzed gibberellins, only GA4, GA9 and GA24 were found in rosemary leaves. GA4 levels showed significant higher levels about 40% for old leaves in control plants compared to young leaves (Figure 3). Interestingly, significant higher levels of GA9, the immediate precursor of GA4, were observed for young leaves of both control and salt-stressed plants compared to old leaves, thus indicating a specific GA9 to GA4 conversion with the induction of leaf senescence in rosemary plants. However, it should be noted that GA4 levels were an order of magnitude lower than those of GA9, thus suggesting that the latter is precursor of different GAs.
Comparing plant hormones contents from Rosmarinus officinalis leaves analyzed by different methods.
UPLC-MS/MS (this study)
Water-stressed plants under controlled conditions
Field-grown plants exposed to summer drought
Salt-stressed plants under controlled conditions
Unlabeled ACC, IPA, Z, ZR, IAA, ABA, JA, SA were purchased from Sigma-Aldrich (Steinheim, Germany). Unlabeled 2iP, DHZ, DHZR, and deuterium labeled d4-ACC, d6-IPA, d5-Z, d5-ZR, d5-IAA, d6-ABA, d4-SA, d5-JA and d6-2iP were purchased from OlChemim Ltd. (Olomouc, Czech Republic). Unlabeled and deuterium labeled gibberellins GA1, GA4, GA9, GA19, GA20, GA24, d2-GA1, d2-GA4, d2-GA9, d2-GA19, d2-GA20, d2-GA24 were purchased from Dr. Lewis Mander at the Australian National University (Canberra, Australia).
Plant material and sampling
All work was carried out with rosemary leaves. Fifteen plants were purchased from a nursery (Vic, Spain) and maintained in a greenhouse at the experimental fields of the University of Barcelona (Barcelona, Spain) with controlled temperature (24/18°C) and adequate water conditions by irrigating the plants with half concentration of Hoagland solution every 2 days. For the optimization procedure of sample preparation and extraction, leaves from these plants were collected, frozen in liquid nitrogen and stored at -80°C until analysis. For the salinity experiment, five plants were exposed to salt stress by irrigating with the same nutrient solution containing an extra addition of 200 mM NaCl, and compared to five control plants (all plants were watered every 2 days). Young and old leaves from the uppermost and lowest part of the plant, respectively, were collected after 8 days of treatment, frozen in liquid nitrogen and stored at -80°C until analyses.
Leaf water content and chlorophyll fluorescence
To determine the relative water content (RWC) of leaf material from the salinity experiment, young and old leaves were collected, immediately weighed (FW), re-hydrated for 24 h at 4°C in darkness (TW) and subsequently oven-dried for 48 h at 60°C (DW). The RWC was determined as 100 × (FW-DW)/(TW-DW). Measurements of the maximum efficiency of photosystem II photochemistry (Fv/Fm ratio) were made by using a pulse-modulated fluorimeter Imaging-PAM (Walz, Effeltrich, Germany) after 2 h of dark adaptation. The F v /F m ratio was calculated as (Fm-F0)/Fm, where Fm and F0 are the maximum and basal fluorescence yields, respectively, of dark-adapted leaves.
Frozen leaf material (about 100 mg FW.) was ground in liquid nitrogen with the mixer mill MM400 (Retsch GmbH, Haan, Germany) in a 2 ml Eppendorf tube, and then extracted with 1 ml of extraction solvent (methanol:isopropanol, 20:80 (v/v) with 1% of glacial acetic acid) using ultra sonication (4-7°C). The labeled forms of the compounds d4-SA, d6-ABA, d5-JA, d5-IAA, d2-GA1, d2-GA4, d2-GA9, d2-GA19, d2-GA20, d2-GA24, d4-ACC, d6-2iP, d6-IPA, d5-Z and d5-ZR were added as internal standards. D5-Z and d5-ZR were used as internal standards for DHZ and DHZR, respectively. After centrifugation (10,000 rpm for 15 min at 4°C), the supernatant was collected and the pellet was re-extracted with 0.5 ml of extraction solvent and the extraction repeated three times again. Then, supernatants were combined and dried completely under a nitrogen stream and re-dissolved in 300 μl of methanol, centrifuged (10,000 rpm for 5 min) and filtered through a 0.22 μm PTFE filter (Waters, Milford, MA, USA). Samples (5 μl) were then analyzed by UPLC/ESI-MS/MS. Hormones were determined in ten independent samples for each treatment. Quantification was done by the creation of calibration curves including each of the 17 unlabeled analyte compounds (SA, ABA, JA, IAA, GA1, GA4, GA9, GA19, GA20, GA24, ACC, 2iP, IPA, Z, ZR, DHZ and DHZR). Ten standard solutions were prepared ranging from 0.05 to 1000 ng ml-1 and for each solution a constant amount of internal standard (as described above) was added. Calibration curves for each analyte were generated using Analyst™ software (Applied Biosystems, Inc., California, USA). The limit of detection (LOD, S/N = 3) and the limit of quantification (LOQ, S/N = 10) were also calculated with the aid of this software.
The UPLC system consisted of an Aquity UPLC™ System (Waters, Milford, MA USA) quaternary pump equipped with an autosampler. For the analysis of the extracts, a HALO™ C18 (Advanced Materials Technology, Inc., Wilmington, USA) column (2.1 × 75 mm, 2.7 μm) was used. Gradient elution was done with water and 0.05% glacial acetic acid (solvent A) and acetonitrile with 0.05% glacial acetic acid (solvent B) at a constant flow rate of 0.6 ml min-1. Cytokinins and ACC were analyzed using method 1 (M1) and ABA, JA, SA, IAA, and gibberellins were analyzed using method 2 (M2). The gradient profile for M1 (cytokinins and ACC) was applied as follow (t (min), % A): (0, 99), (2, 0), (2.40, 0), (2.60, 99), (3, 99). The gradient profile for M2 (ABA, JA, SA, IAA, and gibberellins) was applied as follow: (t (min), % A): (0, 99), (2.20, 0), (2.40, 0), (2.60, 99), (3, 99). MS and MS/MS experiments were performed on an API 3000 triple quadrupole mass spectrometer (PE Sciex, Concord, Ont., Canada). Analyses for M1 were performed using Turbo Ionspray source in positive ion mode and for M2 in negative ion mode. For both methods temperature was 400°C, nebulizer gas (N2) 10 (arbitrary units), curtain gas (N2) 12 (arbitrary units), collision gas (N2) 4 (arbitrary units) and the capillary voltage was 3.5 kV for M1 and -3.5 kV for M2, respectively. The optimized MS/MS conditions for the analysis of plant hormones are summarized in Additional File 4 and were determined in infusion experiments: a standard solution of each plant hormone and deuterium labeled plant hormone was infused of a constant flow rate of 15 μl min-1 into the mass spectrometer using a Model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). The mass spectrometer was operated in multiple reaction mode (MRM) due to their high selectivity using precursor-to-product ion transitions because many compounds could present the same nominal molecular mass or peaks can overlap. Since more than 100 GAs with partly same molecular masses and similar retention times are found in plants special mention is needed for GAs identification in plant extracts. Additional files 5 and 6 show fragmentation patterns for labeled and unlabeled GA1, GA4, GA9, GA19, GA20, GA24 standards. In rosemary leaf extracts GA4, GA9 and GA24 were detected. Using multiple reaction monitoring (MRM) conditions a specific precursor to one product ion transition is monitored. However, to verify the identification of GAs in rosemary leaf extracts the specific precursor ions of GA4, GA9 and GA24 to two different product ions in MRM mode were monitored. All GAs mass chromatograms from rosemary leaf extracts showed identical retention times as GA standards.
Differences between treatments were evaluated using the analysis of variance (ANOVA), using the DMS's post hoc test, and were considered significant at a probability level of P < 0.05.
Support for the research was received through grants BFU2006-01127, BFU2009-07294-E, BFU2009-06045, and CSD2008-00040 from the Ministry of Science and Innovation of the Spanish Government, and the ICREA Academia prize to SMB funded by the Generalitat de Catalunya. The authors wish to thank all lab members (M.E. Abreu, I. Hernández, M.A. Asensi-Fabado, J. Cela, M. Oñate and L. Arrom) for their help in method optimization during the last 5 years. We are also especially indebted to the technical staff of the Serveis Científico-Tècnics of the University of Barcelona, and particularly O. Jáuregui for their help in method optimization.
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