- Open Access
A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress
© Forcat et al; licensee BioMed Central Ltd. 2008
- Received: 08 February 2008
- Accepted: 30 June 2008
- Published: 30 June 2008
We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue.
- Salicylic Acid
- Jasmonic Acid
- Multiple Reaction Monitoring
- Tomato DC3000
- Jasmonic Acid Level
Phytohormones play an important role in mediating host responses to various biotic and abiotic stresses such as pathogen challenge, insect herbivory, drought, cold and heat stress. Traditionally, salicylic acid (SA) and jasmonic acid (JA) have been, respectively, associated with resistance to biotrophic and necrotrophic pathogens (reviewed in [1, 2]. Although classical SA and JA responsive molecular markers indicate that these phytohormones function antagonistically, recent studies suggest that both the timing and amplitude of hormonal signals play key roles in determining the final pathological phenotype [3, 4].
Emerging evidence suggests that a key strategy of plant pathogens is to modify plant hormone levels to promote pathogenicity. Consequently, pathogens have evolved complex repertoires of effector proteins whose functions include modulation of basal phytohormone levels during development of disease. For example, during foliar infection, the hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, delivers ~30 effector proteins into the plant cell . Experimental data suggest they act with a surprising degree of redundancy to modify host signalling pathways, and one clear strategy is to suppress or modify plant hormone responses [6, 7].
Recently, the stress hormone, abscisic acid (ABA), better known for its role in response to drought stress and maintenance of seed dormancy (reviewed by (8) has been demonstrated to influence plant pathogen interactions [9–12]. Emerging evidence suggests there are most likely antagonistic interactions between ABA and, JA/ET (ethylene)  or SA, signalling pathways depending upon the lifestyle of the infecting pathogen. Thus it is important to be able to measure changes in endogenous concentrations of these hormones at different stages of the infection process. Moreover there is an increasing interest in crosstalk between biotic and abiotic stress pathways , how plants prioritize their responses under a given stress and how plants respond to multiple stresses. Plants clearly use phytohormonal signals in a combinatorial manner to achieve distinct outcomes yet actual levels of individual hormones are seldom measured, and if so, only a single hormone is usually quantitated. However the evidence for perturbation of one hormone pathway can having profound effects on synthesis and accumulation of other hormones is considerable . Conventional methods for measuring the hormones such as using enzyme-linked immuno-sorbent assay (ELISA), high-performance liquid chromatography (HPLC) or gas chromatography/mass spectrometry (MS) methods are of limited sensitivity or require a lengthy derivatisation process. Recently we have used C18 solid phase extraction columns for reliable measurements of the acidic hormones SA, ABA and JA [10, 16], however the methodology is time-consuming. Currently, no single method appears to be suitable for the range of hormones implicated in plant pathogen interactions.
We therefore sought to develop a robust quantitative analysis using crude soluble plant extracts by exploiting the high sensitivity of LC/MS. Here we present a method for determining ABA, JA and SA from a single extract that is rapid, accurate, technically simple and requires minimal amounts of tissue. The nature of the method lends itself to high throughput phytohormone determination from time-delimited sampling of plant responses in which these hormones are suspected to participate. This method provides several advantages over previously published methods which individually measure ABA, JA and SA [17–19] as these approaches require time-consuming additional steps such as partitioning of the extracts, solvent evaporation by the use of a rotary evaporator, drying of the sample under N2 and resuspension of the residue. Such manipulations compromise the speed of the process, increase potential technical error and restrict its use as a high throughput method. Moreover, this is also the first report where these three acidic hormones are accurately measured from a single extract.
While, plant hormones such as ABA has been measured individually in crude extracts,  no one method has been published that allows simultaneous simple, rapid and accurate measurement of the three acidic hormones, JA, SA and ABA via LC/MS. We therefore developed a method with an extraction solvent that allowed the reproducible and stable extraction of the analytes of interest from relatively small amounts of starting material as well as the ability to inject directly relatively large volumes of the sample whilst retaining good peak shapes. While here we report characterization of this method on Arabidopsis thaliana leaves, this method is equally applicable to other plant species such as tomato (M. Grant unpublished).
Plants were grown for four to five weeks in a controlled environment chamber under short days (10 h), 70% humidity as previously described .
Pathogen or abiotic stressed plant material was harvested into liquid nitrogen and freeze dried. Samples were next placed in a 2 ml microfuge tube and ground in a bead beater (Qiagen or equivalent) with 3 mm tungsten beads at 25 Hz/s for 3 min. Ten milligram of powdered tissue (~110 mg fresh weight, or equivalent to approximately two fully expanded Arabidopsis leaves) was weighed into a new 2 ml microfuge tube and extracted with 400 μl of 10% methanol containing 1% acetic acid to which internal standards had been added (1 ng of 2H6 ABA, 10 ng of 2H2 JA and 13.8 ng 2H4 SA). Each treatment also included an extraction control containing no plant material. A 3 mm tungsten bead was placed in each microfuge tube and samples were extracted in the bead beater for 2 min at 25 Hz/s, placed on ice for 30 min then centrifuged at 13,000 g for 10 min at 4°C. The supernatant was carefully removed and the pellet re-extracted with 400 μl of 10% methanol containing 1% acetic acid. Following a further 30 min incubation on ice the extract was centrifuged and the supernatants pooled. The two extractions resulted in 90–95% recovery of the targeted analytes.
Samples (50 μl) were then analysed by HPLC-electrospray ionisation/MS-MS using an Agilent 1100 HPLC coupled to an Applied Biosystems Q-TRAP 2000 (Applied Biosystems, California, USA). Chromatographic separation was carried out on a Phenomenex Luna 3 μm C18(2) 100 mm × 2.0 mm column, at 35°C. The solvent gradient used was 100%A (94.9% H2O: 5% CH3CN: 0.1% CHOOH) to 100%B (5% H2O: 94.9% CH3CN: 0.1% CHOOH) over 20 min. Solvent B was held at 100% for 5 min then the solvent returned to 100% A for 10 min equilibration prior to the next injection. The solvent flow rate was 200 μl/min. To reduce contamination of the MS, the first 2 min of the run was directed to waste using the inbuilt Valco valve.
Analysis of the compounds was based on appropriate Multiple Reaction Monitoring (MRM) of ion pairs for labelled and endogenous JA, SA and ABA using the following mass transitions; 2H2-JA 211 > 61, JA 209 > 59, 2H4 SA 141 > 97, SA 137 > 93, 2H6 ABA 269 > 159, ABA 263 > 153, SA-glyc 299 > 93.
The MS was operated in the negative mode using Turbo-Ionspray™ as the ion source. Optimal conditions were determined using the Quantitative Optimisation feature of the Analyst software both by infusing standards into the MS by syringe pump and injecting standards into a 200 μl/min flow of 50% Solvent A/50% Solvent B.
The optimised conditions were as follows: Temperature 400°C, Ion source gas 1 50 psi, Ion source gas 2 60 psi, Ion spray voltage -4500 V, curtain gas 40 psi, CAD gas setting 2; the DP (-25 V), EP (-9) and CEP (-2) were held constant for all transitions. Collision energies (CE) and dwell times (DT) were specific for each compound/internal standard pair, the parameters used were JA CE-25, DT 100 ms, ABA CE-17, DT 250 ms and SA CE-38, DT 50 ms. Data were acquired and analysed using Analyst 1.4.2 software (Applied Biosystems).
Hormones were determined in three independent samples for each treatment or timepoint.
Reproducibility of the phytohormone extraction method
Hormone extraction reproducibility in technical replicate of extracts of wounded and desiccated tissue.
Reproducibility of LC/MS measurements
LC-MS reproducibility in hormone determination following 10 replicate injections of a stress-treated extract
RSD % Quantification
RSD % Retention Time
Phytohormones remain stable two days after extraction.
RSD % Quantification
RSD % Retention Time
Linearity of response
ABA levels were induced by leaving a detached leaf to desiccate at room temperature (~22°C, 60% relative humidity) for 2 h. ABA levels were determined relative to adjacent attached leaves (Fig. 5b). ABA levels are generally undetectable in leaves of Arabidopsis plants grown under controlled environmental conditions unless specially adapted methods are used. By contrast, 2 h of desiccation caused an ~800% increase in ABA levels. The LOD for ABA was ~4 fold that obtained by Lopez-Carbonella & Jaureugi (2005). Their protocol used two different organic extractions and an optimised HPLC method to target ABA. Given the simplicity of our extraction protocol and added ability to detect JA and SA this LOD compares favourably.
Changes in endogenous SA levels were demonstrated by comparing pathogen challenged control plants with the isochorismate synthase 1 deficient plant (sid2). Col-0 and sid2 plants were inoculated with either virulent P. syringae pv. tomato DC3000 (DC3000) or the type three secretion deficient DC3000 hrpA mutant (21). Salicylic acid was determined 21 h post inoculation. As expected, in wild type plants both DC3000 and the hrp mutant accumulate significant amounts of SA and SA-glycoside, whereas levels of these metabolites were strongly attenuated in the sid2 background (Fig. 5c). By contrast, both ABA (Fig 5d) and JA (data not shown) levels increased following challenge with DC3000 as previously determined using 70% methanol extracts and C18 solid phase extraction columns prior to LC/MS .
We have developed a rapid, high throughput, cost effective method for quantification of the three major stress hormones in Arabidopsis. The method requires minimal tissue, is highly reproducible and can accurately measure phytohormones across the expected physiological dynamic range. Moreover, it compares well with other methods that have more complex extraction methods that specifically target the individual hormones, ABA, JA or SA targeted here. The use of freeze dried material promotes ease of handling and automation. Yield decreases associated with freeze drying compared to fresh-frozen material, probably due either to analyte insolubilisation or to volatilization during the freeze drying process, were minimized. This method is equally applicable to fresh or freeze dried tissues and the experimental circumstances will dictate the starting material. In our experience, use of freeze dried tissue is more convenient for scaling up extraction, especially when weighing multiple samples, e.g. during for time course analyses. The advantage of using Multiple Reaction Monitoring is that it is relatively easy to customise runs to identify other discriminatory metabolites, such as aromatic derived secondary compounds, which are readily associated with plant stress responses.
We would like to thanks Wendy Byrne for excellent technical support. This work was funded through a British Biotechnology and Science Research Council grant BB/D007046/1 to MG and JWM.
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