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Physicochemical Quantification of Abscisic Acid Levels in Plant Tissues with an Added Internal Standard by Ultra-Performance Liquid Chromatography
通过添加内标的超高效液相色谱法对植物组织中的脱落酸水平进行组织化学定量测定   

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Abstract

The phytohormone abscisic acid (ABA) is critical for a range of plant responses to the environment, most importantly in closing the stomata of seed plants during drought (Mittelheuser and Van Steveninck 1969; Brodribb et al. 2014). The high precision quantification of this hormone by physicochemical methods is a relatively simple process, essential for studies that aim to investigate the role or action of this hormone in plants. Outlined here is a method for the extraction, purification and quantification of ABA levels in plant tissues. This method involves methanolic extraction of ABA from homogenised tissue. Purification of ABA is then undertaken by a simple etheric partitioning method. A focus is placed on determining ABA levels in leaves; however this method is suitable for all tissue types, including plant solutes such as xylem sap, seeds (both dry and green) and large samples of tissue such as root systems.

Keywords: Plant hormones(植物激素), Abscisic acid(脱落酸), Stomata(气孔), Chromatography(色谱法)

Materials and Reagents

  1. Methanol (Merck Schuchardt OHG, catalog number: 603-001-00-X )
  2. 2, 6-di-tert-butyl-4-methylphenol (BHT) (Sigma-Aldrich, catalog number: B1378 )
  3. Liquid nitrogen (optional for manual homogenization method only)
  4. Labelled or deuterated ABA (such as 13C labelled, [2H6] ABA or other form with any number of deuterium atoms) (e.g. OlChemIm Ltd, catalog numbers: 03 2721-03 2723 ; catalog numbers: 35671-08-0 )
  5. Acetic acid (Analytical grade, any supplier)
  6. Diethyl ether (Analytical grade, any supplier)
  7. Acetonitrile (for UPLC-MS) (Sigma-Aldrich, catalog number: 271004 )
  8. Aluminium foil
  9. 50 ml conical centrifuge tube (Greiner Bio-One GmbH, catalog number: 210261 )
  10. 0.5 ml Eppendorf tubes (any brand will suffice)
  11. 2 ml screw-cap tubes (Scientific Specialities, catalog number: 2330-00 ) with tethered screw tube caps (Scientific Specialities, catalog number: 2002-59 ) (optional for very small samples or samples that require long-distance freight)
  12. Whatman no. 1 filter paper (optional for large samples only)
  13. 1.5 ml Eppendorf tubes (any brand will suffice)
  14. Centrifuge for 2 ml Eppendorf tubes
  15. Filtered glass 150 mm Pasteur pipette s (Poulten & Graf GmbH, model: D810 )
  16. Rubber nipple for Pasteur pipette
  17. 10-100 µl pipette (any brand will suffice)
  18. 100-1,000 µl pipette (any brand will suffice)
  19. 5 ml pipette and tips (any brand will suffice)
  20. 80% methanol in water (v/v) (see Recipes)
  21. 2% acetic acid (v/v) (see Recipes)
  22. 80% methanol in water (v/v) with added butylated hydroxytoluene (BHT) (see Recipes)
  23. 80% methanol in water (v/v) (see Recipes)
  24. 2% acetic acid (v/v) (see Recipes)
  25. 5% methanol, 94% water and 1 % acetic acid (v/v) (see Recipes)
  26. 1% acetic acid (v/v) (see Recipes)

Equipment

  1. Balance (±0.001 g)
  2. Scholander pressure chamber (e.g. PMS Instrument Company, model: 600 ) (optional for specialised experiments or quantification of ABA from xylem sap)
  3. Stainless steel beads 7 mm (QIAGEN, catalog number: 69989 / 69990 ) (optional for very small samples only)
  4. Modelling clay or Blu-tak (Bostik) (optional for very small samples only)
  5. Glass beaker (variable size) (optional for large samples)
  6. General purpose 21 cm scissors
  7. Tissue homogeniser [either modified Ba-mix® or Physcotron (Microtec Co., model: NS-7 )]
  8. Cell lysis machine (e.g. QIAGEN, model: TissueLyser II ) (optional for very small samples only)
  9. Conventional Ba-mix® or similar hand-held blender (optional for large samples only)
  10. Ceramic mortar and pestle (optional for manual homogenization method only)
  11. Büchner funnel (optional for large samples only)
  12. Vacuum flask (optional for large samples only)
  13. Vacuum sample concentrator (or rotary evaporator with 100 ml round bottom flask, optional for large samples)
  14. Fume hood
  15. Heating block that can contain 0.5 ml Eppendorf tubes (gentle stream of nitrogen gas, passing through a low flow regulator, tubing and funnelled through a standard 100 µl pipette tip, if desired) (e.g. Ratek Instruments, model: DBH10DP )
  16. A LCGC Certified Clear Glass 12 x 32 mm Screw Neck Vial, with Cap and PTFE/silicone Septum , 2 ml Volume, 100 /pkg [(Waters, catalog number: 186000272C ), Insert 150 µl with preinstalled plastic spring (Waters, catalog number: WAT094171 )]
  17. Aerosol barrier pipette tips for both µl pipettes (these are important to prevent sample contamination of the pipette which can compromise future samples. They can be made by inserting a small amount of cotton wool into the barrel end of the pipette tip.) (any brand will suffice)
  18. Ultra-performance liquid chromatograph and multiple reaction-monitoring tandem mass spectrometer [Water Acquity H-series UPLC coupled to a Waters Xervo triple quadrupole mass spectrometer, containing a Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm x 1.7 µm particles)]

Software

  1. Waters MassLynx and TargetLynx software

Procedure

  1. For leaf samples: A 50 ml conical centrifuge tube should be pre-labelled and stabilised on the weighing platform of a ±0.001 g balance by being placed in a glass beaker. Approximately 0.05-0.5 g of leaf tissue (as required or necessary, depending on the amount of tissue available for the species) was harvested, weighed on the balance into the conical centrifuge tube. Tissue collected should reflect the experiment undertaken, but for most experiments the newest, fully expanded leaf is preferable.
    Note for step 1: It is important that the balance, beaker and labelled conical centrifuge tube are tared prior to sampling to save inadvertent and unnecessary exposure of tissue to desiccation in the air post excision. Generally 0.05-0.1 g of tissue is more than adequate, however much lower weights of tissue can be measured, for example 0.01 g of epidermal tissue with high resolution (McAdam and Brodribb, 2015). When determining the amount of tissue to be harvested, consideration of the expected level of ABA in the tissue (drought stressed plants will have a high abundance of ABA and thus less tissue maybe required), the amount of secondary metabolites, resins or mucilage in the species of interest (less tissue maybe better in species with an abundance of either or all of these compounds e.g. conifers). In large angiosperm leaves, a sample from the middle of the leaf, or region of interest, is all that is required; for imbricate conifers, twigs suffice; in general petioles and stems though, should be avoided when sampling for foliar ABA. See Figure 1.
    For xylem sap samples: Tissue for which ABA analysis is required should be enclosed in a Scholander pressure chamber and an over pressure of 0.4 MPa applied to the sample, the first drop should be discarded due to contamination from cytosolic contents after which the sap can be collected into an 0.5 ml Eppendorf tube, continue from step 6. See Figure 2.
    For very small samples (less than 0.05 g) of species with very soft leaves/tissue (e.g. angiosperm herbs, some ferns and Selaginella species or leaf epidermis) or samples of green seeds: Tissue can be harvested and weighed (±0.001 g) into 2 ml screw-cap tubes containing a stainless steel bead that has been pre-labelled, stabilised on the weighing platform of the balance in a small piece of modelling clay or Blu-Tack and tared prior to harvesting.
    For very large samples (e.g. whole root systems or entire large leaves, samples > 0.7 g): Tissue can be harvested and weighed (±0.001 g) into a glass beaker of appropriate size, pre-labelled and tared on the balance prior to harvesting.


    Figure 1. Representative leaves of angiosperms (top panel) and a lycophyte, fern and conifer (bottom panel) with an example of the tissue taken to quantify ABA from each (shown in the images immediately below). Note that a leaflet of Pisum sativum can be used, while a section of leaf from Olea europaea, Eucalyptus globulus and Persea americana are used. Similarly in Selaginella kraussiana a small section of stem with leaves, pieces of pinnules of the fern Blechnum nudum and small branch of the conifer Callitris rhomboidea can be used.


    Figure 2. An example of xylem sap being collected from a sample enclosed in a Scholander pressure chamber. The collection tube can be held in place with Blu-Tack or modeller’s clay.

  2. For leaf samples: The sample was then chopped into smaller pieces using scissors and covered in approximately 8-10 ml of cold (-20 °C) 80% methanol in water with added BHT (see Figure 3 for an example of the extent to which samples should be chopped at this stage).
    For samples in 2 ml screw-cap tubes the sample should just be covered in approximately 1.5 ml of cold 80% methanol in water with added BHT (see Figure 4 for an example of tissue enclosed in 2 ml screw-cap tubes at this stage).
    For very large samples: The sample was chopped into smaller pieces using scissors and covered with cold 80% methanol in water with added BHT.


    Figure 3. An example of the extent to which samples of the conifer Callitris rhomboidea (right) and angiosperm Olea europaea (left) should be chopped immediately prior to covering with cold 80% methanol in water with added BHT. The same samples (not to scale) are shown both in 50 ml conical centrifuge tubes covered with cold 80% methanol in water with added BHT (top) and on filter paper (bottom) (not to scale). Scale bar for filter paper images is shown below.


    Figure 4. Leaflet tissue of the angiosperm Pisum sativum (left) (taken from Figure 1) and a green seed of Pisum sativum (right) enclosed in 2 ml screw-top tubes and covered in cold 80% methanol with added BHT. Note the stainless steel bead that has already been added to the tube at the bottom, and the correct tethering of the tube (a step critical for successful long-distance freight).

  3. Samples were then stored at -20 °C overnight to ensure that all biochemical processes in the tissue were deactivated.
    Notes for steps 1-3: As ABA levels in the leaves of angiosperms can increase rapidly on exposure to dry air or dehydration (McAdam and Brodribb, 2015) it is critical that as little time as possible is taken from harvesting tissue to covering the sample in cold 80% methanol in water with added BHT. It is especially important to prevent the samples from being exposed to the open air or sunlight after excision [as isomerisation of ABA can rapidly take place (Brabham and Biggs, 1981)], so wrapping tissue in damp paper towelling, then aluminium foil, and enclosing wrapped tissue in a plastic zip-lock bag and placing in a dark box is critical if any number of samples are being collected at the same time. So long as leaves do not desiccate, leaf water potential can be quantified using a Scholander pressure chamber. For this samples must be wrapped in damp paper towelling, aluminium foil and bagged to avoid desiccation. The Scholander pressure chamber should also contain damp paper towelling. If these steps are adhered to prior to weighing the sample for ABA analysis, the quantification of ABA will not be adversely affected (Brodribb et al., 2014). Gradual increases and decreases in pressure should, however, always be applied to the tissue with care being taken to avoid over pressurisation, for correct use see ‘Pressure Chamber’ PrometheusWiki http://prometheuswiki.publish.csiro.au/tiki-pagehistory.php?page=Pressure%20chamber&preview=14.
    Notes for step 3 regarding samples collected remotely: For samples collected where access to a freezer is not immediately available, after weighing, samples can be stored on ice in the dark until such time as a freezer is available. Provided that the tissue has been placed at -20 °C overnight, it is then possible to transport samples at room temperature (in the dark) so long as all of the samples in an experiment are exposed to the same conditions prior to step 5.
    Notes for step 3 regarding samples requiring long-distance freight: For samples that are collected a substantial distance from the laboratory where purification, extraction and quantification will take place, requiring long-distance freight for transport, tissue should be collected, briskly chopped and weighed into screw-cap tubes similar to that described for very small samples, but without the stainless steel beads. Samples should be covered in cold 80% methanol in water with added BHT and stored at -20 °C overnight. It is then possible to transport these samples by air freight. Care must be taken to ensure that the lids of the screw-cap tubes are correctly sealed to avoid loss of sample due to changes in pressure (see Figure 4). Standard (non-screw-capped) Eppendorf tubes should never be used for samples that will be transported by long-distance freight.
  4. For leaf samples: Tissue was homogenized using a Physcotron [or modified Ba-mix® (Figure 5)]. Care should be taken when rinsing the homogenizer to not increase the volume of the sample excessively (i.e. the volume should below 20 ml, ideally 10 ml, for increased accuracy), for this 80% methanol in water (v/v) at room temperature should be used. See Figure 6 for an example of the extent of homogenization expected for the tissue shown in Figure 3 after using a Physcotron.
    For very small samples in screw-cap tubes containing a stainless steel bead: Tissue was homogenized using a cell lysis machine run until samples are ground well (approximately 2 min). See Figure 7.
    For samples in screw-cap tubes that have been transported by long-distance freight: tissue and solutes should be transferred to 50 ml conical centrifuge tubes and homogenised using a Physcotron (or modified Ba-mix®). It is important to thoroughly rinse the tubes using 80% methanol in water.
    For very large samples in a beaker: Tissue was homogenized using a conventional un-modified Ba-mix® or other hand-held blender of appropriate size for the beaker. Care should be taken to rinse the Ba-mix® after homogenization with 80% methanol in water (v/v) into the sample.
    Special note for step 4: In the absence of all other means of tissue homogenization, samples can be harvested as described in step 1, wrapped in aluminium foil and immersed in liquid nitrogen. Samples can then be stored at -70 °C until homogenization is required. Homogenization of such samples can be undertaken using a mortar and pestle, pre-cooled by filling mortar with liquid nitrogen and which should be allowed to evaporate. Tissue should be initially broken gently with the pestle then, before defrosting, ground to a fine powder in a small volume of liquid nitrogen. All of this powder should be then briskly transferred while cold to a 50 ml conical centrifuge tube, weighed (as described in step 1) and covered in cold 80% methanol in water with added BHT, it is especially important to ensure that this is done briskly so as to not allow de-frosting of the sample, more liquid nitrogen should be added if this is occurring. Special care should be taken when working with liquid nitrogen.
    Following homogenization proceed immediately to step 5.


    Figure 5. Modified Ba-Mix® for homogenizing plant tissues in a 50 ml centrifuge tube. Note the movable aluminium sleave, the right-hand image shows the sleave fully raised exposing the modified blade. The blade of this Ba-Mix® spins at high speed and, if the sleeve is moved up and down while homogenizing, the sample is forced through the spinning blade and is macerated. It is important that the tissue is forced passed the blade by adjusting the height of the sleeve relative to the blade.


    Figure 6. An example of the degree of homogenization after the use of a Physcotron on the samples of the conifer Callitris rhomboidea (left) and angiosperm Olea europaea (right) shown in Figure 4. Note the increase in liquid from rinsing the homogenizer in 80% methanol in water after use.


    Figure 7. An example of the homogenization obtained after the use of cell lysis machine and stainless steel bead on the leaflet and green seed samples shown in Figure 4. Note that this method is only effective for soft, herbaceous leaves or green seeds.

  5. To each sample, 15 ng of deuterated [2H6] ABA (or similar) was added.
  6. ABA was then extracted from the homogenized tissue by passive diffusion into the 80% methanol in water with added BHT by incubating at 4 °C, overnight.
    Note for step 6: After this step samples can be stored for extended periods of time, in the dark at any temperature (although preferably at -20 °C).
  7. For all samples in a 50 ml conical centrifuge tube: An aliquot of 5 ml was taken from each sample, being careful not to disturb the settled pellet of homogenized tissue in the tube.
    For very small samples in 2 ml screw-cap tubes: Tubes were centrifuged for 5 min at 13,000 x g and a 1 ml aliquot was taken from each sample, being careful not to disturb the pellet.
    For very large samples: Samples were vacuum filtrated through a Büchner funnel and Whatman no 1 filter paper. An aliquot of at least 50% of this filtrate was taken from each sample.
  8. The aliquot was dried under vacuum at no more than 30 °C.
    Note on step 8: For 5 ml of sample drying times using a sample concentrator normal take 5 h, 1 ml of sample for very small samples approximately 1 h, large volumes from very large samples can be dried using a rotary evaporator and normally take approximately 15 to 30 min, depending on the volume.
  9. ABA was then resuspended in 500 µl of 2% acetic acid in water (v/v) by pipetting and transferred to a 1.5 ml Eppendorf tube.
  10. 300 µl of diethyl ether was added to each tube and the tube was inverted vigorously a number of times to ensure the partitioning of ABA into the ether phase. Steps using diethyl ether should be undertaken in a fumehood. If inversion results in emulsion, a brief centrifugation (1 min at 13,000 x g) will result in the separation of the two distinct phases.
  11. Using a glass Pasture pipette, the diethyl ether phase (top phase) was collected and placed into a 500 µl Eppendorf tube. See Figure 8 for differentiating the diethyl ether layer.


    Figure 8. The visual differentiation of the diethyl ether phase containing ABA above the 2% acetic acid in water phase is apparent. Holding tubes at a slight angle, as depicted, greatly aids the differentiation of these layers for ease of pipetting.

  12. Steps 10 and 11 were repeated, with the second diethyl ether phase pooled with the first.
    Note for steps 11 and 12: Care should be taken to avoid collecting any of the 2% acetic acid in water layer when collecting the diethyl ether phase.
  13. Samples in diethyl ether were then dried to completeness on a heating block at 40 °C (to decrease drying times samples can be dried under a nitrogen stream).
  14. After drying, ABA was resuspended in 150 µl of 5% methanol in 2% acetic acid in water (v/v) (this volume can be reduced according to the expected level of ABA in the sample; samples with lower levels of ABA may need to be resuspended in only 50 µl).
  15. Tubes were then centrifuged at the maximum speed for a bench-top centrifuge at room temperature for 3 min, to ensure that any particulate matter form a pellet (i.e. 13,000 x g).
  16. 50 µl from each sample was taken, avoiding any pelleted particulate matter, and placed into an appropriate autosampling vial for UPLC-MS analysis.
    Note for step 16: samples should be covered in aluminium foil to protect from sunlight.
  17. Detailed UPLC-MS analysis methods can be found in McAdam and Brodribb (2012).
    Briefly the method is was as follows:
    Solvents used were 1% acetic acid (v/v) in water (A) and acetonitrile (B), a flow rate 0.35 ml/min was used with a gradient of 80% A: 20% B to 5% A: 95% B at 5 min, equilibrated to starting conditions for 3 min. Column temperature was 45 °C and injection volume 40 µl. The mass spectrometer was operated in negative ion electrospray mode. Needle and cone voltages were 2.7 kV and 32 V respectively. Selected reaction monitoring was used to detect endogenous ABA and [2H6]ABA. The ion source and desolvation temperature were 130 °C and 450 °C respectively. The desolvation gas and cone gas was nitrogen at flow rates of 950 L/h and 50 L/h respectively. Tandem MS transitions monitored for ABA were mass to charge ratios (m/z) 263.2 to 153.1, 204.2 and 219.2, for [2H6]ABA the transitions were 269.2 to 159.1, 207.2 and 225.2. Collision energies for m/z 263.2 to 153.1 and 204.2 were 18 V, for m/z 263.2 to 219.2 was 16 V, this was similar for the corresponding deuterium-labelled channels. Dwell time was 50 ms for each channel. Data were analysed using the Waters MassLynx and TargetLynx software. Only the m/z 263.2 to 153.1 and corresponding deuterium-labelled channels were used for quantification.
  18. To quantify ABA levels in the sample (in terms of fresh weight) the following formula was used:


    Example A: A 0.0783 g leaf sample of the fern Blechnum nudum (shown in Figure 1) was harvested and prepared as described above for leaf samples. The resulting chromatogram from the UPLC-MS of this sample is depicted in Figure 9. As 15 ng of labelled [2H6]-ABA was added to the sample during preparation the level of endogenous ABA in the sample was calculated as follows:





    Figure 9. An example raw chromatogram output from the UPLC-MS analysis of a leaf sample from the fern Blechnum nudum showing the peaks of [2H6]ABA (top) and endogenous ABA (bottom). Above the peaks are shown the retention time (left, this is also depicted as the x-axis of the graph) and ion intensity (right, shown as relative ion intensity on the y-axis).

    Example B: Unstressed foliar ABA levels vary depending on the species. Table 1 provides mean foliar ABA levels from a range of unstressed species.

    Table 1. Mean foliar ABA levels in unstressed individuals from across the diversity of vascular land plants
    Species
    Family
    Mean unstressed foliar ABA level (ng g-1 FW)
    Reference
    Selaginella kraussiana
    Selaginellaceae
    11.4 ±1.4
    McAdam and Brodribb, 2012
    Blechnum nudum
    Blechnaceae
    1.505
    Example A
    Callitris rhomboidea
    Cupressaceae
    402 ±60
    Brodribb and McAdam, 2013
    Pinus radiata
    Pinaceae
    104 ±10
    Brodribb and McAdam, 2013
    Quercus robur
    Fagaceae
    17.5 ±1.3
    McAdam and Brodribb, 2015
    Pisum sativum
    Fabaceae
    3.85 ±1.1
    McAdam and Brodribb, 2015

Recipes

  1. 80% methanol in water (v/v) (1 L)
    Combine 200 ml of dH2O with 800 ml of methanol
    Stored at room temperature in glassware, provided it is not allowed to evaporate, this reagent can be used indefinitely.
  2. 2% acetic acid (v/v) (100 ml)
    Combine 2 ml of glacial acetic acid with 98 ml of dH2O
    Stored at room temperature in glassware this reagent can be used indefinitely
  3. 80% methanol in water (v/v) with added butylated hydroxytoluene (BHT) (1 L)
    Dissolve 250 mg of BHT in an aliquot of methanol
    Add remaining methanol (total methanol, including dissolving aliquot, should be 800 ml)
    Add 200 ml of dH2O
    Store and use at -20 °C
    This reagent can be used directly from the stock, it should be stored in glassware and once mixed, provided it is not allowed to evaporate can be used indefinitely.
  4. 80% methanol in water (v/v) (1 L)
    Combine 200 ml of dH2O with 800 ml of methanol
    Stored at room temperature in glassware, provided it is not allowed to evaporate, this reagent can be used indefinitely.
  5. 2% acetic acid (v/v) (100 ml)
    Combine 2 ml of glacial acetic acid with 98 ml of dH2O
    Stored at room temperature in glassware this reagent can be used indefinitely
  6. 5% methanol, 94% water and 1 % acetic acid (v/v) (100 ml)
    Combine 5 ml of methanol, 94 ml of dH2O and 1 ml of glacial acetic acid
    Stored at room temperature in glassware, provided it is not allowed to evaporate, this reagent can be used indefinitely.
  7. 1% acetic acid (v/v) (100 ml)
    Combine 1 ml of glacial acetic acid with 99 ml of dH2O
    Stored at room temperature in glassware this reagent can be used indefinitely

Acknowledgements

This protocol was adapted from previously published studies McAdam and Brodribb (2012) and McAdam and Brodribb (2014) and was performed by McAdam and Brodribb (2015). This work was supported by Australian Research Council grants. Thanks must go to John Ross for invaluable advice on improving methods of sample preparation over many years and the Central Science Laboratory, University of Tasmania for undertaking UPLC-MS analyses and method development.

References

  1. Brabham, D. E. and Biggs, R. H. (1981). Cis-trans photoisomerization of abscisic acid. Photochem Photobiol 34: 33-37.
  2. Brodribb, T. J. and McAdam, S. A. (2013). Abscisic acid mediates a divergence in the drought response of two conifers. Plant Physiol 162(3): 1370-1377.
  3. Brodribb, T. J., McAdam, S. A., Jordan, G. J. and Martins, S. C. (2014). Conifer species adapt to low-rainfall climates by following one of two divergent pathways. Proc Natl Acad Sci U S A 111(40): 14489-14493.
  4. McAdam, S. A. and Brodribb, T. J. (2012). Fern and lycophyte guard cells do not respond to endogenous abscisic acid. Plant Cell 24(4): 1510-1521.
  5. McAdam, S. A. and Brodribb, T. J. (2014). Separating active and passive influences on stomatal control of transpiration. Plant Physiol 164(4): 1578-1586.
  6. McAdam, S. A. and Brodribb, T. J. (2015). The evolution of mechanisms driving the stomatal response to vapor pressure deficit. Plant Physiol 167(3): 833-843.
  7. Mittelheuser, C. J. and Van Steveninck, R. F. M. (1969). Stomatal closure and inhibition of transpiration induced by (RS)-abscisic acid. Nature 221:281-282.

简介

植物激素脱落酸(ABA)对于一系列植物对环境的反应至关重要,最重要的是在干旱期间闭合种子植物的气孔(Mittelheuser和Van Steveninck 1969; Brodribb等人2014) 。 通过物理化学方法对这种激素的高精度定量是一个相对简单的过程,对于旨在研究该激素在植物中的作用或作用的研究是必要的。 这里概述了用于提取,纯化和定量植物组织中ABA水平的方法。 该方法包括从匀浆组织中甲醇提取ABA。 然后通过简单的以太分区方法进行ABA的纯化。 重点放在确定叶中的ABA水平; 然而该方法适用于所有组织类型,包括植物溶质如木质部汁液,种子(干的和绿色的)和组织的大样品例如根系统。

关键字:植物激素, 脱落酸, 气孔, 色谱法

材料和试剂

  1. 甲醇(Merck Schuchardt OHG,目录号:603-001-00-X)
  2. 2,6-二叔丁基-4-甲基苯酚(BHT)(Sigma-Aldrich,目录号:B1378)
  3. 液氮(仅适用于手动均化方法)
  4. 标记的或氘化的ABA(例如具有任何数目的氘原子的标记的[2 H] 6 H 6 ABA或其他形式) em>例如 OlChemIm Ltd,目录号:03 2721-03 2723;目录号:35671-08-0)
  5. 乙酸(分析级,任何供应商)
  6. 二乙醚(分析级,任何供应商)
  7. 乙腈(用于UPLC-MS)(Sigma-Aldrich,目录号:271004)
  8. 铝箔
  9. 50ml锥形离心管(Greiner Bio-One GmbH,目录号:210261)
  10. 0.5 ml Eppendorf管(任何品牌都足够)
  11. 2ml螺旋盖管(Scientific Specialties,目录号:2330-00) 带有栓塞螺纹管帽(Scientific Specialties,目录号: 2002-59)(非常小的样品或需要的样品可选) 长途货运)
  12. Whatman没有。 1滤纸(仅适用于大样品)
  13. 1.5 ml Eppendorf管(任何品牌都足够)
  14. 离心2ml Eppendorf管
  15. 过滤玻璃150mm巴斯德移液管(Poulten& Graf GmbH,型号:D810)
  16. 巴斯德吸管的橡胶奶嘴
  17. 10-100μl移液器(任何品牌都足够)
  18. 100-1,000μl移液器(任何品牌都足够)
  19. 5毫升移液器和提示(任何品牌都足够了)
  20. 80%甲醇水溶液(v/v)(参见配方)
  21. 2%乙酸(v/v)(参见配方)
  22. 添加丁基化羟基甲苯(BHT)的80%甲醇水溶液(v/v)(见配方)
  23. 80%甲醇水溶液(v/v)(参见配方)
  24. 2%乙酸(v/v)(参见配方)
  25. 5%甲醇,94%水和1%乙酸(v/v)(参见配方)
  26. 1%乙酸(v/v)(参见配方)

设备

  1. 平衡(±0.001g)
  2. Scholander压力室(例如PMS仪器公司,型号:600)(对于专门实验或对来自木质部汁液的ABA的定量)是可选的
  3. 不锈钢珠7 mm(QIAGEN,目录号:69989/69990)(仅适用于非常小的样品)
  4. 建模粘土或Blu-tak(Bostik)(仅适用于非常小的样本)
  5. 玻璃烧杯(可变尺寸)(大样品可选)
  6. 通用21厘米剪刀
  7. 组织匀浆器[改良的Ba-mix或Physcotron(Microtec Co.,型号:NS-7)]
  8. 细胞裂解机(例如,QIAGEN,型号:TissueLyser II)(仅对非常小的样品可选)
  9. 常规Ba-mix ®或类似的手持搅拌器(仅适用于大样本)
  10. 陶瓷砂浆和杵(仅适用于手动均化方法)
  11. Büchner漏斗(仅适用于大样品)
  12. 真空瓶(仅适用于大型样品)
  13. 真空样品浓缩器(或带100 ml圆底烧瓶的旋转蒸发器,可选大样品)
  14. 通风橱
  15. 加热块可以包含0.5ml Eppendorf管(温和的氮气流,通过低流量调节器,管道和漏斗通过标准的100μl移液管吸头,如果需要)(例如,Ratek Instruments,型号:DBH10DP)
  16. LCGC认证的透明玻璃12 x 32毫米螺旋颈瓶,带帽和PTFE /硅胶隔膜,2毫升体积,100/pkg [(Waters,目录号:186000272C),插入150μl预装塑料弹簧:WAT094171)]
  17. 气雾屏障移液器吸头的两个移液管(这些是重要的,以防止样品污染的移液器,可以损害未来的样品。他们可以通过插入少量的棉花棉球的吸管尖端的桶端)(任何品牌就足够了)
  18. 超高效液相色谱和多反应监测串联质谱仪[Water Acquity H-series UPLC,与Waters Xervo三重四极杆质谱联用,含有Waters Acquity UPLC BEH C18柱(2.1mm×100mm×1.7μm颗粒)]

软件

  1. Waters MassLynx和TargetLynx软件

程序

  1. 对于叶片样品:应将50ml圆锥形离心管置于玻璃烧杯中,在±0.001g天平的称重平台上预先标记并稳定。收获约0.05-0.5g叶组织(根据需要或必要,取决于可用于该物种的组织的量),在天平上称重到锥形离心管中。收集的组织应反映进行的实验,但对于大多数实验,最新的,完全扩张的叶是优选的 对于步骤1的注意:重要的是,在取样之前对天平,烧杯和标记的圆锥形离心管进行去皮,以便在切除后空气中将组织无意和不必要地暴露于干燥。通常0.05-0.1g的组织是足够的,然而可以测量低得多的组织重量,例如0.01g具有高分辨率的表皮组织(McAdam和Brodribb,2015)。当确定要收获的组织的量时,考虑组织中ABA的预期水平(干旱胁迫的植物将具有高丰度的ABA,因此可能需要更少的组织),次生代谢物,树脂或胶浆在感兴趣的物种(较少的组织在具有这些化合物中的任一种或所有的丰度的物种中可能更好,例如针叶树)。在大被子植物叶中,来自叶的中间或感兴趣区域的样品是所有需要的;对于针叶树,树枝就足够了;通常叶柄和茎,在叶面ABA取样时应避免。见图1。
    对于木质部汁液样品:需要ABA分析的组织应该封闭在Scholander压力室中,并且施加到样品的0.4MPa的超压,第一滴应当由于来自胞质内容物的污染而被丢弃,在此之后,收集到0.5ml Eppendorf管中,从第6步继续。见图2 对于具有非常柔软的叶/组织(例如被子植物药草,一些蕨类植物和卷柏种类或叶表皮)的非常小的样品(小于0.05g)或绿色样品种子:可以收获组织并称重(±0.001g)到含有不锈钢珠的2ml螺旋盖管中,所述不锈钢珠已经预先标记,在天平的称重平台上稳定在一小块模型粘土或Blu-在收获前粘和和去皮。
    对于非常大的样品(例如全根系或整个大叶,样品> 0.7g):可以收获组织并称重(±0.001g)到适当尺寸的玻璃烧杯中,预先标记并在收获前平衡天平

    图1.被子植物的代表性叶子(上图)和乳糜泻,蕨类植物和针叶树(下图),其中具有用于定量来自每一个的ABA的组织的实例(显示在下面的图像中)。注意,可以使用豌豆豌豆的小叶,同时可以使用来自欧洲桉属(Olea europaea),桉树(Eucalyptus globulus)和美洲猕猴桃/em>。类似地,在卷柏(Selaginella kraussiana)中,具有叶的茎的一小部分,蕨类植物的小苞片和针叶树的小分支 Callitris rhomboidea 可以使用。


    图2.从封闭在Scholander压力室中的样品收集的木质部汁液的实例。收集管可以用Blu-Tack或模型粘土保持在适当位置。

  2. 对于叶片样品:然后用剪刀将样品切成较小的片,并在加入BHT的约8-10ml冷(-20℃)80%甲醇水溶液中覆盖(参见图3, 在此阶段应切碎样品)。
    对于2ml螺旋盖管中的样品,应该将样品刚好覆盖在大约1.5ml的加有BHT的冷的80%甲醇水溶液中(关于在该阶段封装在2ml螺旋盖管中的组织的实例,参见图4) 。
    对于非常大的样品:用剪刀将样品切成较小的块,并用加有BHT的冷的80%甲醇水溶液覆盖。


    图3.应当在覆盖之前立即切碎针叶树样品(右)和被子植物(Olea europaea)(左)的样品的程度的实例用冷的80%甲醇的水溶液加入BHT。 在用加有BHT(顶部)和滤纸(底部)(未按比例)的冷的80%甲醇水覆盖的50ml锥形离心管中显示相同的样品(不按比例)。滤纸图像的比例尺如下所示。


    图4.被子植物豌豆豌豆(左图)(取自图1)和豌豆豌豆的绿色种子(右)的叶片组织,其封闭在2ml螺旋盖管,并且在加有BHT的冷80%甲醇中覆盖。注意已经添加到底部管中的不锈钢珠,以及管的正确栓系(成功长的关键步骤 - 运费)。

  3. 然后将样品在-20℃下储存过夜,以确保组织中的所有生化过程失活 对于步骤1-3的注释:由于被子植物的叶中的ABA水平在暴露于干燥空气或脱水时可以快速增加(McAdam和Brodribb,2015),因此尽可能少的时间是从收获组织用加有BHT的冷的80%甲醇水溶液覆盖样品。防止样品在切除后暴露于露天或阳光下是特别重要的[由于ABA的异构化可以迅速发生(Brabham和Biggs,1981)],因此将包装纸置于湿纸巾中,然后是铝箔,将包裹的组织包裹在塑料拉链袋中并放置在暗箱中是关键的,如果在同一时间收集任何数量的样品 时间。只要叶片不干燥,叶片水势可以使用Scholander压力室量化。对于这些样品必须用潮湿纸巾包裹,铝箔和袋装以避免干燥。 Scholander压力室还应包含湿纸巾。如果在称量样品用于ABA分析之前遵循这些步骤,则ABA的定量不会受到不利影响(Brodribb等,2014)。然而,压力的逐渐增加和减少总是应用于组织,小心避免过度加压,正确使用请参见"压力腔"PrometheusWiki http://prometheuswiki.publish.csiro.au/tiki-pagehistory.php?page=Pressure% 20chamber& preview = 14。
    步骤3关于远程收集的样品的注意事项:对于在不能立即获得冷冻器的情况下收集的样品,称重后,样品可以在黑暗中的冰上储存,直到获得冷冻箱。如果组织已经置于-20℃过夜,则可以在室温(在黑暗中)运输样品,只要实验中的所有样品在步骤5之前暴露于相同的条件。
    对于需要长途运输的样品,步骤3的注意事项:对于需要进行纯化,提取和定量的实验室,需要长距离货物运输的实验室收集的样品,应该快速收集组织切碎并称重到螺旋盖管中,类似于对于非常小的样品描述的,但没有不锈钢珠。样品应该在加有BHT的冷的80%甲醇水溶液中覆盖并在-20℃下储存过夜。然后可以通过空运来运输这些样品。必须注意确保螺旋盖管的盖子正确密封,以避免由于压力变化而导致的样品损失(参见图4)。标准(非螺旋盖)Eppendorf管不应用于将通过长途运输运输的样品。
  4. 对于叶样品:使用Physcotron [或改良的Ba-mix(图5)]将组织匀浆。当冲洗均化器时,应注意不要过度增加样品的体积(为了提高精确度,体积应低于20ml,理想的是10ml),对于该80%的甲醇水溶液v/v)。参见图6,示出了在使用Physcotron之后对于图3所示的组织预期的均匀化程度的示例。
    对于含有不锈钢珠的螺旋盖管中的非常小的样品:使用细胞裂解机使组织均化,直到样品被充分研磨(约2分钟)。见图7.
    对于已通过长距离运输运输的螺旋盖管中的样品:将组织和溶质转移至50ml锥形离心管中并使用Physcotron(或改良的Ba-mix)均化。重要的是用80%甲醇水溶液彻底冲洗试管。
    对于烧杯中的非常大的样品:使用用于烧杯的适当尺寸的常规未改性的Ba-mix或其他手持式混合器将组织匀浆。在样品中用80%甲醇水溶液(v/v)匀浆后,应小心冲洗Ba-mix 。
    步骤4的特别注意:在没有所有其他组织匀浆方法的情况下,可以如步骤1所述收获样品,包裹在铝箔中并浸入液氮中。然后将样品储存在-70℃下,直到需要均质化。这样的样品的均化可以使用研钵和杵进行,通过用液氮填充砂浆预冷却并且应该允许其蒸发。组织应该最初用杵捣碎,然后,在除霜之前,在小体积的液氮中研磨成细粉末。然后将所有这些粉末在冷却下迅速转移到50ml锥形离心管中,称重(如步骤1所述),并且在加入BHT的冷的80%甲醇水溶液中覆盖,特别重要的是确保这样做以便不允许样品脱霜,如果发生这种情况,应该加入更多的液氮。使用液氮时应特别小心。
    均化后立即进入步骤5

    图5.修改Ba-Mix ®,用于在50 ml离心管中匀浆植物组织。注意可移动的铝制凹槽,右侧图像显示了完全凸起暴露改性叶片。这种Ba-Mix的刀片以高速旋转,并且如果套筒在均匀化的同时上下移动,则样品被迫通过旋转刀片并被浸软。重要的是,通过调节套筒相对于刀片的高度,迫使组织通过刀片

    图6.使用Physcotron对针叶树Callitris rhomboidea(左)和被子植物Olea europaea的样品(右)的同质化程度的实例)。注意,使用后,在80%甲醇的水溶液中漂洗均化器后液体的增加。。


    图7.使用细胞裂解机和不锈钢珠在小叶和绿色种子样品上获得的均质化的实例,如图4所示。请注意,该方法仅对软的,草本的叶或绿色种子
  5. 向每个样品中加入15ng的氘化的[2 H] H 6 6 ABA(或类似的)。
  6. 然后通过在4℃下孵育过夜通过被动扩散进入80%甲醇水溶液并加入BHT从匀浆组织中提取ABA。
    注意步骤6:此步骤后,样品可以在任何温度下在黑暗中储存较长时间(但最好在-20°C)。
  7. 对于在50ml锥形离心管中的所有样品:从每个样品中取出5ml的等分试样,小心不要扰乱管中均匀化组织的沉降的沉淀。
    对于2ml螺旋盖管中的非常小的样品:将管在13,000×g离心5分钟,从每个样品中取1ml等分试样,小心不要打扰沉淀。 > 对于非常大的样品:将样品通过Büchner漏斗和Whatman no 1滤纸真空过滤。从每个样品中取出至少50%的该滤液的等分试样
  8. 将等分试样在不超过30℃的真空下干燥 关于步骤8的注意:对于使用样品浓缩器的5ml样品干燥时间,通常需要5小时,1ml非常小的样品的样品约1小时,来自非常大的样品的大体积可以使用旋转蒸发器干燥, 通常需要约15至30分钟,具体取决于音量。
  9. 然后通过移液将ABA重新悬浮在500μl2%乙酸水溶液(v/v)中,并转移到1.5ml Eppendorf管中。
  10. 向每个管中加入300μl乙醚,并将管剧烈反转多次,以确保ABA分配到乙醚相中。使用乙醚的步骤应在通风柜中进行。如果反转导致乳液,短暂离心(13,000×g下1分钟)将导致两个不同阶段的分离。
  11. 使用玻璃牧场移液管,收集二乙醚相(上相)并放入500μlEppendorf管中。参见图8,用于区分乙醚层

    图8.含有ABA的水相中的2%乙酸以上的乙醚相的视觉分化是显而易见的。如图所示,以小角度保持管极大地有助于这些层的分化容易移液。

  12. 重复步骤10和11,用第一个二乙醚相合并 注意步骤11和12:收集乙醚相时应注意避免收集水层中的任何2%乙酸。
  13. 然后将二乙醚中的样品在40℃的加热块上干燥至完全(以减少干燥时间,样品可在氮气流下干燥)。
  14. 干燥后,将ABA重悬于150μl在2%乙酸水溶液(v/v)中的5%甲醇中(该体积可以根据样品中ABA的预期水平降低;具有较低水平的ABA的样品可能需要重悬于50μl)。
  15. 然后将试管以最大速度在室温下在台式离心机中离心3分钟,以确保任何颗粒物质形成沉淀(即,13,000xg )。
  16. 从每个样品取50μl,避免任何颗粒状颗粒物质,并放入适当的自动取样小瓶进行UPLC-MS分析。 请注意步骤16:样品应覆盖铝箔,以防阳光。
  17. 详细的UPLC-MS分析方法可以在McAdam和Brodribb(2012)中找到 简单的方法如下:
    使用的溶剂是在水(A)和乙腈(B)中的1%乙酸(v/v),使用流速0.35ml/min,梯度为80%A:20%B至5%A :95%B,5分钟,平衡至起始条件3分钟。柱温度为45℃,注射体积为40μl。质谱仪在负离子电喷雾模式下操作。针和锥电压分别为2.7 kV和32 V.选择的反应监测用于检测内源性ABA和[ ] ABA。离子源和去溶剂化温度分别为130℃和450℃。脱溶剂气体和锥形气体分别为950L/h和50L/h的流量的氮气。对于ABA监测的串联MS跃迁的质荷比(m/z)263.2至153.1,204.2和219.2,对于[ 2 /em> 6 ] ABA的转换是269.2到159.1,207.2和225.2。 m/z 263.2至153.1和204.2的碰撞能量为18V,对于m/z 263.2至219.2为16V,这对于相应的氘标记的通道是类似的。每个通道的驻留时间为50 ms。使用Waters MassLynx和TargetLynx软件分析数据。只有m/z 263.2至153.1和相应的氘标记通道用于定量。
  18. 为了定量样品中的ABA水平(以鲜重计),使用以下公式:


    实施例A:收获0.0783g蕨菜的叶片样品(如图1所示),并如上所述制备叶片样品。从该样品的UPLC-MS得到的色谱图示于图9中。在制备期间向样品中加入15ng标记的[2 H] H 6 6] -ABA样品中内源性ABA的水平计算如下:





    图9.来自蕨菜的叶样品的UPLC-MS分析的示例性原始色谱图输出,显示了来自the>>的峰, 6] ABA(顶部)和内源性ABA(底部)。以上 峰显示保留时间(左,这也被描绘为图的x轴)和离子强度(右,显示为y轴上的相对离子强度)。

    实施例B:未胁迫叶面ABA水平根据种类而变化。 表1提供了一系列无应变物种的平均叶面ABA水平
    表1.来自血管性土地植物多样性的未受胁迫个体的平均叶面ABA水平
    种类
    家庭
    平均无胁迫叶面ABA水平(ng g -1 FW)
    参考
    Selaginella kraussiana
    卷柏科
    11.4±1.4
    McAdam和Brodribb,2012年
    Blechnum nudum
    白桦科 1.505
    示例A
    Callitris rhomboidea
    柏木科
    402±60
    Brodribb和McAdam,2013年
    辐射
    松属
    104±10
    Brodribb和McAdam,2013年
    栎树
    Fagaceae
    17.5±1.3
    McAdam和Brodribb,2015年
    豌豆荚
    豆科 3.85±1.1·
    McAdam和Brodribb,2015年

食谱

  1. 80%甲醇水溶液(v/v)(1L) 将200ml dH 2 O与800ml甲醇混合 在室温下储存在玻璃器皿中,只要不能蒸发,该试剂可以无限期使用。
  2. 2%乙酸(v/v)(100ml) 将2ml冰醋酸与98ml dH 2 O混合 在室温下储存在玻璃器皿中,此试剂可无限期使用
  3. 80%甲醇水溶液(v/v)中加入丁基化羟基甲苯(BHT)(1L) 将250mg BHT溶解在等分试样的甲醇中
    加入剩余的甲醇(总甲醇,包括溶解等分试样,应为800ml) 加入200ml dH 2 O
    在-20°C下存储和使用
    这种试剂可以直接从原料中使用,应该储存在玻璃器皿中,一旦混合,只要不允许蒸发即可无限期使用。
  4. 80%甲醇水溶液(v/v)(1L) 将200ml dH 2 O与800ml甲醇混合 在室温下储存在玻璃器皿中,只要不能蒸发,该试剂可以无限期使用。
  5. 2%乙酸(v/v)(100ml) 将2ml冰醋酸与98ml dH 2 O混合 在室温下储存在玻璃器皿中,此试剂可无限期使用
  6. 5%甲醇,94%水和1%乙酸(v/v)(100ml) 合并5ml甲醇,94ml dH 2 O和1ml冰醋酸。 在室温下储存在玻璃器皿中,只要不能蒸发,该试剂可以无限期使用。
  7. 1%乙酸(v/v)(100ml) 将1ml冰醋酸与99ml dH 2 O混合 在室温下储存在玻璃器皿中,此试剂可无限期使用

致谢

该协议改编自以前发表的研究McAdam和Brodribb(2012)和McAdam和Brodribb(2014),由McAdam和Brodribb(2015)进行。这项工作得到澳大利亚研究委员会资助。感谢您对John Ross多年来改进样品制备方法的宝贵意见,以及塔斯马尼亚大学中央科学实验室进行UPLC-MS分析和方法开发的宝贵意见。

参考文献

  1. Brabham,D.E。和Biggs,R.H。(1981)。 反式光致异构化脱落。 Photochem Photobiol 34:33-37。
  2. Brodribb,T.J。和McAdam,S.A。(2013)。 脱落酸介导两种针叶树干旱反应的差异。植物Physiol 162(3):1370-1377。
  3. Brodribb,T.J.,McAdam,S.A.,Jordan,G.J.and Martins,S.C。(2014)。 针叶树物种通过遵循两个不同的途径之一适应低降雨量的气候。 em> Proc Natl Acad Sci USA 111(40):14489-14493。
  4. McAdam,S.A。和Brodribb,T.J。(2012)。 蕨类植物和lycophyte保卫细胞对内源性脱落酸没有反应。植物Cell 24(4):1510-1521。
  5. McAdam,S.A。和Brodribb,T.J。(2014)。 分离主动和被动对蒸腾气孔控制的影响。植物生理 164(4):1578-1586。
  6. McAdam,S.A.和Brodribb,T.J。(2015)。 驱动气孔反应对蒸气压力缺乏的机制的演变。植物Physiol 167(3):833-843
  7. Mittelheuser,C.J。和Van Steveninck,R.F.M。(1969)。 气孔闭合和抑制(RS) - 脱落酸诱导的蒸腾。 221:281-282
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. McAdam, S. A. (2015). Physicochemical Quantification of Abscisic Acid Levels in Plant Tissues with an Added Internal Standard by Ultra-Performance Liquid Chromatography. Bio-protocol 5(18): e1599. DOI: 10.21769/BioProtoc.1599.
  2. McAdam, S. A. and Brodribb, T. J. (2014). Separating active and passive influences on stomatal control of transpiration. Plant Physiol 164(4): 1578-1586.
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