Quantification of Abscisic Acid, Cytokinin, and Auxin Content in Salt-Stressed Plant Tissues

Abstract

Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope with the stress, e.g., suppression of growth or mobilization of plant metabolism. This chapter describes a procedure enabling such quantification. Due to the high variability of these hormones in plant tissues, it is advantageous to determine their content in the same sample. Reverse phase and ion exchange chromatography allows separation of the individual hormone fractions. Hormones as well as their metabolites can be identified and quantified by LC/MS.

1 Introduction

Plant interactions with the often adverse environment are, at least partially, regulated by phytohormones. The hormone analysis can provide important information about the physiological state of plants and can contribute to the prediction of their further behavior.

The key hormone in the defence to abiotic stresses, including salinity, is abscisic acid (ABA). ABA mediates both fast responses (modulation of ion flows resulting in stomata closure) and relatively longer-term changes in the expression of many stress-associated genes. Regulation of stomata aperture is crucial to cope with water deficit. Re-arrangement of transcriptome results in stimulation of the formation of a range of defence molecules, e.g., dehydrins. ABA-regulated genes represent over 10% of the genome in Arabidopsis seedlings (1). ABA is a sesquiterpenoid (15-carbon). According to the position of carboxyl group at side chain, cis- and trans-isomer can be distinguished, the former one exhibiting the biological activity. Due to the optically active C1′, bearing a hydroxyl group, ABA may occur either as (S)/(+) isomer, which is the most physiologically active, or as much less active (R)/(−) isomer. ABA can be deactivated by conjugation to glucose. ABA glucosylester and glucoside are important ABA storage forms. ABA can be irreversibly inactivated to phaseic, dihydrophaseic, or neophaseic acid.

Recent reports suggest that apart from the stimulation of the defence mechanisms, an important part of the stress response is a modulation of plant growth and development. Cell division is governed predominantly by two hormones—auxins and cytokinins. The active endogenous auxin is indole-3-acetic acid (IAA), which stimulates cell division and elongation, as well as promotes apical dominance, positively affects root growth, mediates tropic responses, and prevents abscision (2). Vast amount of IAA (90–95%) is present in plants in a conjugated form, mostly with amino acids (aspartate, leucine, alanine, or glutamine) or sugars. These conjugates represent either storage or deactivation forms. IAA can be also metabolized to indole-butyric acid.

Cytokinins are defined as substances stimulating (at the presence of auxin) cell division (cytokinesis) (3). They are indispensable for cell cycle transition at both check points—G₂/M and G₁/S (4). They positively affect photosynthesis (5), delay senescence, and enhance the sink strength (6). They play a critical role in balancing acquisition and distribution of macronutrients (7). Natural cytokinins are adenine derivatives with either isoprenoid or aromatic side chain. The physiologically active forms are cytokinin bases (predominantly trans-zeatin) and, to lesser extent, cytokinin ribosides. Cytokinins are deactivated either by cleavage of the side chain with cytokinin oxidase/dehydrogenase or by conjugation with glucose. Glucosylation of hydroxyl at the side chain results in reversible cytokinin O-glucosides (storage forms), glucosylation at purine ring in position N9− and N7−, is ­irreversible. Cytokinin phosphates (nucleotides) are immediate precursors of bioactive cytokinins. In some cases, their level correlates with the rate of cytokinin biosynthesis. They can be, however, also products of cytokinin back conversion, especially after sudden increase of the level of bioactive cytokinins (e.g., after application of exogenous cytokinin).

Cytokinins and ABA exhibit antagonistic effects on regulation of many processes involved in stress responses (cell division, stomata aperture, photosynthetic activity). Their ratio may reflect plant strategies to cope with stress. As the defence is highly energy demanding, suppression of growth, coinciding with high ABA and low cytokinin levels, might allow redirection of the (limited) energy sources to the defence. On the other hand, stabilization of photosynthesis, as the energy source, by maintenance of relatively high cytokinin levels could lead to mobilization of plant metabolism. This may be advantageous, especially in case of milder or short stresses.

Estimation of phytohormone content in plants is challenging due to their very low concentrations (in the range of picomoles per gram of fresh weight (FW)). At the same time, plant tissues are very rich in diverse classes of potential interferences, such as primary and secondary metabolites that are present at much higher concentrations (usually micro- to millimoles per gram FW). The first techniques used for the estimation of hormone content in plant tissues were biotests. Subsequent development of analytical methods enabled more specific determination of phytohormones. In the early days of hormone analysis, the quantification techniques had rather low selectivity and sensitivity. Thus, hormone analysis required large amounts of material (usually several grams) and involved several comprehensive purification steps. The whole procedure was rather demanding, very labor-, time-, and material-consuming.

Progress in hormone analyses is associated with the development of antibodies specific to individual hormones (8). Quantification of the hormone concentration in the sample was based on the competition for a limited amount of antibodies, either with radiolabeled standard (radioimmunoassay) or with standards coupled to the enzyme (e.g., phosphatase or peroxidase, enzyme immunoassay). The limitations of immunotechniques have been specificity of antibodies, as well as the presence of interfering substances in the sample. Due to relatively small size of hormone molecules, antibodies can be raised only to the conjugate of the hormone and high molecular “carrier,” usually BSA. In consequence, antibodies have rather low recognition for the part of the molecule used for binding to the protein. Immunological analysis is still commonly used in case of ABA, as the structure of deactivation products differ sufficiently from the active molecule, but it could be quite misleading in case of cytokinins. The highly abundant cytokinin deactivation products, cytokinin N-glucosides, can exhibit quite significant cross-reactivity. Therefore, cytokinin fractionation by high performance liquid chromatography (HPLC) and subsequent immunological determination of cytokinins in the individual fractions is used. This approach is complicated by potential variation in retention times, caused by other compounds present in the sample (amounts of which highly prevail that of hormones) or by their effect on hormone-antibody interaction (which could be both negative as well as positive).

Recent advancements of analytical instrumentation allowed significant increase of efficiency, selectivity, sensitivity, and throughput of phytohormone analyses. The method of choice for phytohormone analysis is HPLC coupled to mass spectrometric (MS) detector. Modern HPLC can separate efficiently and quickly complex mixtures of compounds with a wide range of polarity without need of derivatization. Modern mass spectrometers have three features that make them almost ideal detector: sensitivity, selectivity, and speed. MS is one of the most sensitive types of detectors capable of detecting a few femtomoles of an analyte. The sensitivity of MS allows one to decrease significantly the amount of material needed for analysis to as little as tens to a hundred milligrams FW. This simplifies sample collection procedures and allows analysis of materials in limited amount, such as individual plant parts or tissues of small plants. Selectivity of MS is based on its intrinsic capability to measure the very specific parameter of a compound, its mass. Selecting and monitoring of a particular mass filters out all other masses. The recorded chromatogram contains only compounds with the same mass, isomers, or isobars (i.e., nonrelated compounds with the same weight). Even more selective is the tandem MS, also known as MS/MS. It contains two MS with a collision cell in between. The first MS allows the passage of one mass (precursor) that enters the collision cell, where it is broken into compound-specific fragments. The fragments then enter the second MS where they are filtered and only pre-selected fragments are allowed to pass through to the detector. Only compounds with pre-selected precursor-to-fragment mass transitions are detected. Tandem MS is so selective, that often chromatogram of crude extract can contain just one peak of the target compound. The superior selectivity of MS increases the confidence in compound identity and allows simplification of purification procedure to just one or a few steps. Recent progress in electronics of MS has made their responses very short, in the range of few milliseconds, allowing rapid measurement and switching between many pre-set masses. The MS speed permits simultaneous analysis of tens to hundreds of compounds in a single sample, leading to recent expansion of multi-metabolite profiling, also known as metabolome analysis.

In order to obtain reliable, physiologically relevant data on hormone content, severe precautions need to be taken for the sample collection. All phytohormones exhibit substantial diurnal variations. Thus, approximately the same time of a day should be used for sampling. It should be taken into the consideration that levels of hormones change during the year. Samples taken in the winter would substantially differ from the spring and summer ones (even in the case of plants grown in cultivation chambers without any contact with daylight). Also, the intensity of light (and its spectrum) significantly affects hormone pool, which complicates repetitions of the experiments in different labs. The individual tissues (leaf, root, stem) differ in hormone levels. Quite big differences are also among individual leaves and even within the leaf blade; more actively growing parts have higher cytokinin levels, e.g., the basal part of monocotyledonous leaves.

The procedure for phytohormone analysis includes: (a) sampling, (b) extraction, (c) purification, and (d) quantitative determination using HPLC-MS/MS. The procedure described by Dobrev and Kaminek (9) allows determination of three groups of plant hormones, cytokinins, auxins, and ABA, in a single sample.

2 Materials

2.1 Extraction

• 1. Mortar and pestle.

• 2. Liquid nitrogen (LQN).

• 3. Pipettes (50 μL, 5 mL).

• 4. Measuring cylinders (1 L, 100 mL).

• 5. Analytical balances.

• 6. Methanol, p.a. grade (“per analysis”—purity for chemical analyses).

• 7. Double-distilled water.

• 8. Formic acid, p.a. grade.

• 9. Appropriate stable isotope labeled internal standards (²H₅-tZ, ²H₅-tZR, ²H₅-tZRMP, ²H₅-tZ7G, ²H₅-tZ9G, ²H₅-tZOG, ²H₅-tZROG, ²H₃-DZ, ²H₃-DZR, ²H₃-DZRMP, ²H₃-DZ9G, ²H₇-DZOG, ²H₆-iP, ²H₆-iPR, ²H₆-iPRMP, ²H₆-iP7G, ²H₆-iP9G; ²H₆-ABA, ²H₃-PA, ²H₃-DPA, ²H₄-7OH-ABA, ²H₅-ABA-GE, ¹³C₆-IAA).

• 10. 2 mL Eppendorf tubes for samples <100 mg FW, 50 mL centrifugation tubes for samples >100 mg FW.

• 11. Centrifuge.

• 12. Freezers (−20, −80°C).

2.2 Purification

• 1. Methanol, p.a. grade.

• 2. Formic acid, p.a. grade.

• 3. Acetic acid, p.a. grade.

• 4. Ammonia (Ammonium hydroxide 26%), p.a. grade.

• 5. MilliQ water.

• 6. SPE C18 column, e.g., Sep-Pak Plus, Waters, part # WAT036810.

• 7. SPE Oasis MCX column, 6 cc/150 mg, Waters, part # 186000256.

• 8. SPE Vacuum Manifold, 12 or 24-port, Supelco, part # 57250-U (to mount SPE columns for simultaneous purification of up to 12 or 24 samples).

• 9. SpeedVac, i.e., vaccum evaporator with vacuum pump (in case of oil pump also freezing trap (to −100°C) to collect the evaporating organic solvents).

• 10. Calf-intestine alkaline phosphatase (13 U/mg, Sigma, part # P7640).

2.3 MS Quantification

• 1. Acetic acid, LCMS grade.

• 2. Acetonitrile, LCMS grade.

• 3. Water, MilliQ grade.

• 4. Autosampler vials.

• 5. HPLC column, e.g., Luna C18(2), 3 μm, 150  ×  2 mm, Phenomenex.

• 6. HPLC system, e.g., Ultimate 3000, Dionex.

• 7. MS detector, e.g., 3200 QTRAP LC/MS/MS, ABSciex.

2.4 Solvent and Standard Preparation

• Extraction solvent is composed of methanol/double-distilled water/formic acid  =  15/4/1, v/v/v, keep at −20°C.

• Stable isotope labeled internal standards (100 pmol/50 μL per 1 g FW sample) dilute in 50% methanol in water.

• Load solvent: 1 M formic acid: dilute 37.7 mL of 99% formic acid with double-distilled water to 1,000 mL; pH ∼1.4.

• Elute 1 solvent: 100% methanol.

• Elute 2 solvent: 0.35 M NH₄OH: dilute 2.5 mL of 26% ammonia with 97.5 mL double-distilled water, pH ∼11.

• Elute 3 solvent: to 60 mL MeOH add 2.5 mL of 26% ammonia and adjust to 100 mL with double-distilled water.

• Incubation buffer for alkaline phosphatase reaction: 0.1 M ammonium acetate, pH 10.

• Calf-intestine alkaline phosphatase dissolved in incubation buffer at concentration 0.2 U/20 μL for 1 g FW sample (it should be prepared fresh).

3 Methods

3.1 Sampling

• 1. The plant material for hormone analysis should be cut, weigh precisely (also see Notes 1 and 2), and frozen in liquid nitrogen as quickly as possible; because degradative processes are initiated immediately after plant wounding. The fresh weight of plant material could be within a relatively broad range (0.05–1 g FW). Nonetheless, the precise weight (±1%) for each sample must be recorded. The ideal amount of sample depends on the actual amount of hormone in the sample, which needs to be well above the detection limit. The samples can be stored at −80°C.

• 2. When leaf blades are collected from dicotyledonous plants, the main vein should be removed. This is because many hormones are transported via the vascular system, thus the presence of the main (or other big) vein would substantially affect the result of analysis. When monocotyledonous plants are sampled, the apical third of the leaf should be removed, as the already senescent part of the leaf will differ substantially from the basal part. In all cases, the leaves at the same developmental stage should be compared, when evaluating the effect of salinity. Parallel samples for determination of the ratio between fresh and dry weight should be taken when the results are going to be presented per dry weight basis.

• 3. When roots are sampled from plants grown in soil, the soil particles should be quickly removed by brief rinsing with cold tap water and gentle blotting with the tissue. When a hydroponic system is used, usually the roots can be cut, weigh, and freeze immediately.

3.2 Extraction

The aim of the extraction is quantitative release of target compounds from the plant tissue into an extraction solution, and preservation of their chemical integrity. For quantitative recovery, 5 to 1 volumetric ratio of extraction solvent to sample is used, and the sample is extracted twice. The compound degradation is avoided by working at low temperature with an extraction buffer containing a high proportion of organic solvent and a low pH. Extraction is followed by centrifugation. This removes large undissolved and precipitated biopolymers such as cellulose, large nucleic acids, and proteins.

• 1. Homogenize the frozen material with a mortar and a pestle in liquid nitrogen to a fine powder. Care should be taken to avoid any thawing of the tissue.

• 2. Transfer immediately the homogenized, frozen sample (with liquid nitrogen) into an appropriate centrifugation tube, pre-cooled in liquid nitrogen.

• 3. Wash twice the mortar and pestle with five volumes of extraction buffer (cooled to −20°C) and add the wash to the sample in order to transfer quantitatively the sample to the tube. For example, for 100 mg FW sample, wash with two consecutive 0.25 mL of extraction buffer.

• 4. Add internal standards (100 pmol/1 g FW sample or 50 pmol per smaller samples, see Note 3). Mix.

• 5. Place tubes with samples, extraction solvent, and internal standards in freezer at −20°C for 1 h.

• 6. Centrifuge at 15,000  ×  g for 30 min (at 4°C).

• 7. Transfer supernatant into clean tube.

• 8. Add five volumes of extraction solvent to the pellet and mix.

• 9. Incubate for 30 min at −20°C.

• 10. Centrifuge again and combine the supernatants.

3.3 Purification

The aim of purification is to remove from the extract as much as possible of interfering substances without losing significant amount of target compounds. Two solid phase extraction (SPE) columns are used. The first one, C18, is used as a filter for removal of most of the lipophilic substances, with phytohormones passing through. The second SPE column (MCX) retains hormones that are sequentially eluted with appropriate elute solvents into three fractions containing different types of hormones. The fraction containing cytokinin phosphates is incubated with alkaline phosphatase to convert phosphates into ribosides. This conversion is performed due to the lower sensitivity of LC-MS/MS to cytokinin phosphates.

• 1. Using SPE Vacuum Manifold, condition SPE C18 column by washing it with 5 mL methanol, followed by 5 mL extraction solvent (see Subheading 2.4). Do not run the column dry (see Note 4).

• 2. Pass sample extract through SPE C18 column. Collect flow through and evaporate in SpeedVac at 40°C to about 1/10 of volume.

• 3. Dissolve residue into 1 mL of load solvent (see Subheading 2.4).

• 4. Condition SPE Oasis MCX column by washing it with 5 mL methanol followed by 5 mL load solvent. This column can run dry.

• 5. Apply sample onto SPE Oasis MCX column. Discard flow through.

• 6. Wash column with 5 mL load solvent and discard flow through.

• 7. Apply 5 mL elute 1 solvent to MCX column. Collect flow through. This is fraction 1. It contains phytohormones of neutral and acidic character: auxins and ABA.

• 8. Wash column with 5 mL double-distilled water and discard the flow through.

• 9. Apply 5 mL elute 2 solvent to MCX column. Collect flow through. This is fraction 2. It contains cytokinin phosphates (cytokinin nucleotides).

• 10. Apply 5 mL elute 3 solvent to MCX column. Collect flow through. This is fraction 3 and contains cytokinin bases, ribosides, and glucosides.

• 11. Evaporate all three fractions to dryness in SpeedVac at 40°C. Fractions 1 and 3 are ready for quantitative analysis by HPLC-MS/MS.

• 12. Dissolve fraction 2 in 1 mL incubation buffer for alkaline phosphatase. Add alkaline phosphatase and incubate at 37°C for 2 h. Stop the reaction by adding 20 μL glacial acetic acid. This is de-phosphorylated fraction 2.

• 13. Condition SPE C18 column by washing it with 5 mL methanol, followed by 5 mL water.

• 14. Pass de-phosphorylated fraction 2 through SPE C18 column. Discard flow through.

• 15. Wash column with 5 mL water.

• 16. Apply 5 mL 100% methanol and collect flow through. It contains cytokinin ribosides, the de-phosphorylation products of cytokinin phosphates. Evaporate in SpeedVac at 40°C. The de-phosphorylated fraction 2 is ready for analysis.

3.4 Quantification by HPLC-MS/MS

The purified fractions are applied to HPLC-MS/MS, where the individual compounds are separated. The individual hormones and their metabolites are quantified by comparison of the measured response ratio of endogenous hormone to its internal standard and the ratio of hormone of known concentration to internal standard.

The procedure and parameter settings for HPLC-MS/MS are very much instrument-specific. The optimal parameters should be found for the particular instrument (see Note 5). Thus, only general guidance for HPLC-MS/MS follows.

• 1. Dissolve dried sample into 50 μL 10% acetonitrile in water.

• 2. Centrifuge at 15,000  ×  g for 10 min at 4°C.

• 3. Transfer supernatant into autosampler vial.

• 4. Inject the sample. Depending on the amount of extracted sample, inject into HPLC an aliquot, i.e., 1/2 from 100 mg FW extract or 1/10 from 1 g FW extract.

• 5. HPLC conditions: run gradient of A: 5 mM acetic acid in water and B: 5 mM acetic acid in acetonitrile; from 10 to 50% B in 20 min at flow rate 0.25 mL/min. Flush column at 100% B for 5 min and equilibrate to initial conditions for 10 min.

4 Notes

• 1. Hormone content could be expressed either per fresh weight basis (which is directly measured) or by dry weight basis (which may diminish differences caused by different water content among the leaves). It should be taken into account that the individual leaves differ in their water content, so the correspond­ing ratio of fresh and dry weight should be used.

• 2. Hormone levels reflect very precisely the physiological state of individual plants. A considerable difference in hormone levels can be found between the plants in the experiment and even bigger between the independent experiments. Thus, several experiments (at least three) with replicate samples should be analysed to achieve physiologically relevant data.

• 3. Critical points are precise weighing of sample and addition of exactly known amounts of internal standards (which should be at room temperature), since these data will be used in the final calculation of the hormone content.

• 4. SPE column conditioning is important for their proper operation. Loss of a small portion of the sample is not that critical because added internal standards allow to evaluate the losses. However, the losses must not diminish the final amounts of hormones in the samples below the detection limit.

• 5. MS conditions: electrospray ionization at positive mode is advantageous for cytokinin analysis, at negative mode for the other (acidic) hormones. The most intensive ion is usually used for quantification and the others, for identity confirmation. However, in case of high interference, the other (less intensive) ion without substantial background should be used for quantification. Substances for which the labeled standards are not available are determined using the retention times and the mass spectra of unlabeled standards and the response ratio (labeled/unlabeled) of their closest derivative. The multilevel calibration curve is necessary to estimate the linear range of responses as well as detection limits.

  MS parameters need to be optimized for each hormone, especially, ion spray voltage, declustering potential, collision energy, and precursor to fragment transition.

  Since MS is very sensitive instrument, it is very important to keep it clean. Recommended is usage of solvents with the highest purity, e.g., LC/MS grade. Furthermore, the ion source and entrance of MS, gas filters, and oil of the vacuum pump become dirty with use. This leads to gradual decrease of sensitivity. Therefore, cleaning of the ion source and entrance to MS, and replacement of gas filters and pump oil should be carried out at regular intervals.