Sep 26, 2023

Public workspaceProtocol for abscisic acid (ABA) extraction from plant seeds

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbxnnqlpk/v1
Protocol for abscisic acid (ABA) extraction from plant seeds
  • Jian You Wang1,2,
  • Lamis Berqdar1,
  • Salim Al-Babili1,2
  • 1The BioActives Lab, Center for Desert Agriculture, King Abdullah University of Science and Technology, Saudi Arabia;
  • 2The Plant Science Program, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Saudi Arabia
Lamis Berqdar
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbxnnqlpk/v1
Protocol Citation: Jian You Wang, Lamis Berqdar, Salim Al-Babili 2023. Protocol for abscisic acid (ABA) extraction from plant seeds. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxnnqlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 22, 2023
Last Modified: September 26, 2023
Protocol Integer ID: 88357
Keywords: abscisic acid, plant seeds, LC-MS/MS, plant hormone , plant hormone abscisic acid, extraction from plant seed, protocol for abscisic acid, plant seed, protocol for aba extraction, aba extraction, germination, extraction, liquid chromatography, seed dormancy, analysis for aba quantification, mass spectrometry
Funders Acknowledgements:
King Abdullah University of Science and Technology
Grant ID: CDA neodomestication
Disclaimer
The authors declare no competing interests.
Abstract
The plant hormone abscisic acid (ABA) regulates seed dormancy and germination. Here, we present a protocol for ABA extraction from plant seeds. We describe necessary steps required for preparation and extraction, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis for ABA quantification.
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Guidelines
Author Contributions

S.A.-B. and J.Y.W. conceived the project. J.Y.W., and L.B. conducted experiments. L.B., J.Y.W., and S. A.-B. wrote, reviewed, and edited the protocol.
Competing interests
The authors declare no competing interests.
Materials
List of equipment

  • Calibrated balance,
  • Grinder (Mixer Mill MM 400, RETSCH‱),
  • Sonicator (Branson 5510R-DTH Ultrasonic machine),
  • ultra-centrifuge (Centrifuge 5424 R, EppendorfTM),
  • speedVac (Concentrator Plus, EppendorfTM),
  • 0.2 um filter (Non-Sterile Syringe Filter 13 mm; WhatmanTM UnifloTM),
  • 0.1 mL Micro-insert clear glass tube (fisher scientific),
  • 1.5 mL LCMS vial.
Before start
To prepare before to start:

Prepare Amount2 mL of working solution per sample: 100% Methanol spiked with Amount1 µL D6-ABA stock solution.
Sample preparation
2h 42m
Grind 7-8 seeds in a Safe-Lock 2.0 mL Eppendorf tube with 3-4 beads for Duration00:01:00 – frequency 25-26 Hz.
1m
Weight Amount100 mg fine powder of ground seeds.

Add Amount1 mL of Standard solution in each tube.

Pipetting
Sonicate the samples for Duration00:15:00 .

15m
Centrifuge for Centrifigation14000 rpm, 00:05:00 , then transfer the supernatant to a new 2 mL Eppendorf tube.
5m
Centrifigation
Add another Amount1 mL of Standard solution.

Pipetting
Sonicate Duration00:15:00 .

15m
Centrifuge for Centrifigation14000 rpm, 00:05:00 .

5m
Centrifigation
Transfer the supernatant (Amount2 mL ) to a 2 mL Eppendorf tube.

Dry the supernatant under vacuum by speedVac for Duration02:00:00 .

2h
After evaporating the solvent, either keep the extracted samples at Temperature-20 °C or proceed to the next step.
Re-dissolve the final extract in Amount120 µL of acetonitrile : water [25:75 (v/v)] followed by Duration00:01:00 sonication.

1m
Filter the re-suspended solution through a Thikness0.22 µm filter into 0.1 mL micro-insert 29x5.7 mm clear glass tubes with inserted vials.
Tap the bottle to remove any bubbles.
Sample quantification
7m
ABA quantification is performed by LC-MS/MS using a UHPLC-Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific Altis) machine.
Chromatographic separation is achieved on the Hypersil GOLD C18 Selectivity HPLC Columns (150 × 4.6 mm; 3 μm; fisher scientific) with mobile phases consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, and the following linear gradient (flow rate, 0.5 mL/min): 0–10 min, 15%–100 % B, follow it by washing with 100 % B for Duration00:05:00 and equilibration with 15 % B for Duration00:02:00 .
7m
Wash
Inject Amount10 µL of sample, maintain the column temperature at Temperature35 °C for each run.
Set the MS parameters of Thermo ScientificTM AltisTM as follows:
  • negative mode,
  • ion source of H-ESI,
  • ion spray voltage of 3000 V,
  • sheath gas of 40 arbitrary units,
  • aux gas of 15 arbitrary units,
  • sweep gas of 0 arbitrary units,
  • ion transfer tube gas temperature of Temperature350 °C ,
  • vaporizer temperature of Temperature350 °C ,
  • collision energy of 20 eV,
  • CID gas of 2 mTorr, and
  • full width at half maximum (FWHM) 0.4 Da of Q1/Q3 mass.

Note
The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) were the characteristic MRM transitions (precursor ion → product ion) were 263.2 → 153.1 for ABA; 269.2 → 159.1 for D6-ABA.

Protocol references
References

1. Wang, J. Y., Alseekh, S., Xiao, T., et al. (2021). Multi-omics approaches explain the growth-promoting effect of the apocarotenoid growth regulator zaxinone in rice. Communications Biology, 4(1), 1-11. https://doi.org/10.1038/s42003-021-02740-8

2. Chen, G.-T.E., Wang, J.Y., Votta, C., et al. (2023) Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones. bioRxiv Article 535333, 10.1101/2023.04.03.535333.

3. Jamil, M., Alagoz, Y., Wang, J.Y., et al. (2023). Abscisic acid inhibits germination of Striga seeds and is released by them as a rhizospheric signal providing competitive advantage and supporting host infestation. bioRxiv Article 548005, 10.1101/2023.07.06.548005