ABA and ABA catabolites detection and quantification were performed as reported in Diretto et al. (2020) (link) and Barja et al. (2021) (link). Briefly, 50 mg freeze-dried grounded cane samples were extracted using unbuffered Tris-ethyl acetate as reported before (Welsch et al., 2008 (link)). LC-HRMS was carried out using an Ultimate UHPLC system with a photodiode array detector (Dionex), and a Q-exactive quadrupole Orbitrap mass spectrometry system (Thermo Fisher Scientific; LC-HRMS) equipped with an electrospray ionization (HESI) source, operating in negative ion mode, as previously described (Di Meo et al., 2019 (link)) with the following modifications: with nitrogen as sheath and auxiliary gas set at 35 and 25 units, respectively. The vaporizer and capillary temperatures were set at 280 and 320°C, respectively. The discharge current was set to 4.0 μA and S-lens RF level set at 50. Internal standard-based quantification was carried out using the MS data, while retention times and MS2 fragmentation patterns were used for ABA identification by using authentic reference standards (trans-ABA from OlChemIm and ()-ABA from Sigma, St. Louis, MO, United States).
Trans aba
Manufactured by Merck Group
Sourced in United States
Trans-ABA is a laboratory reagent used for chemical analysis and research. It is a trans-isomer of abscisic acid, a plant hormone involved in various physiological processes. Trans-ABA is commonly used as a biochemical standard and research tool in plant biology and related fields. Its core function is to serve as a reference compound for analytical techniques and experimental studies, without further interpretation of its intended use.
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6 protocols using trans aba
1
ABA Quantification in Sugarcane Samples
2
Stress and Drug Administration in Male Rats
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All Sprague Dawley (SD) rats were male and maintained with free access to food and water on a 12h light/dark cycle (lights on 0700h) at a room temperature of 22±1◦C with 50–60% relative humidity. Rats were handled daily for 5min for at least four days before initiating stress or drug administration. The Animal Resource Center of the University of Science and Technology of China reviewed and approved this study.
For the animals that received an intraperitoneal injection, (±)-cis, trans-ABA (Sigma), and fluoxetine hydrochloride (Sigma) were dissolved in a vehicle of sterile saline solution (0.9% w/v sodium chloride) with dimethyl sulfoxide at a ratio of 1:1 (v/v).
For the animals that received an intraperitoneal injection, (±)-cis, trans-ABA (Sigma), and fluoxetine hydrochloride (Sigma) were dissolved in a vehicle of sterile saline solution (0.9% w/v sodium chloride) with dimethyl sulfoxide at a ratio of 1:1 (v/v).
3
Arabidopsis Growth and Stress Responses
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Arabidopsis thaliana (ecotype Col-0) seeds were sterilized in 70% ethanol and 20% bleach as described previously [66 (link)]. After stratification for 3 days at 4 °C, sterilized seeds were directly sown in half strength (1/2) Murashige and Skoog (MS) medium (Caisson Labs) supplemented with 1% sucrose (pH 5.7) and 0.05% MES (w/v). Seeds were germinated and grown in a growth chamber maintained at 22°C (16-h-light and 8-h-dark cycle, 120 µmol−2s−1 light intensity). The roots and shoots of 10-day–old seedlings, and the roots, stems, rosette leaves, flowers and siliques of mature Arabidopsis plants were used for tissue-specific expression analysis. For salt stress, osmotic stress, or ABA treatment, 7-day-old Arabidopsis seedling were transferred into 1/2 MS liquid medium supplemented with 150 mM NaCl, 300 mM mannitol or 100 µM (±)-cis, trans-ABA (Sigma), and the stress treatments were applied for 24 hr. A fresh medium-only control was conducted in parallel. Samples were rinsed with demineralized water, shoot and root tissues were harvested separately, snap-frozen in liquid N2 and stored at −80 °C until further use.
The qRT-PCR experiments were performed according to previous studies [66 (link)]. All the experiments were repeated at least three times using cDNA prepared from two biological replicates. Primers used in the study are listed inSupplemental Table S8 .
The qRT-PCR experiments were performed according to previous studies [66 (link)]. All the experiments were repeated at least three times using cDNA prepared from two biological replicates. Primers used in the study are listed in
4
Quantitative Analysis of Phytohormones
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Frozen pericarp tissues at five ripening stages were lyophilized and ground to a fine powder. At least three different pericarp pools (biological replicates) were analysed for each genotype. 200 mg was extracted for each replicate as previously described (Welsch et al., 2008 ). LC‐HRMS was carried out using a Finnigan Surveyor Plus HPLC System (Thermo Electron), equipped with a 3 μm Hypersil GOLD C18 reverse‐phase column (150 × 4.6 mm; Thermo Electron) as reported before (Ross et al., 2004 ). Internal standard‐based quantification was carried out using the MS data and the quantification software available in the Xcalibur 2.0 software package (Thermo Fisher Scientific, Bremen, Germany). Retention times and MS2 fragmentation patterns were used for identification by using authentic reference standards (trans‐ABA from OlChemIm and (±)‐ABA from Sigma, St. Louis, MO, USA).
5
Rice Stress Response Protocol
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For heavy metal or salt stress, 2-week-old rice seedlings growing in N6 liquid medium were transferred into another N6 solution containing 200 μM Cu2+ (CuSO4) or 200 mM NaCl, respectively; For UV light stress, 2-week-old rice seedlings growing in N6 liquid medium were shifted with dishes together to incubator illuminating with 100 μmol m− 2 s− 1 ultraviolet light; For pathogen stress, Magnaporthe grisea was incubated into PDA (Potato Dextrose Agar) medium growing to the concentration of 3 × 105 spore ml− 1 and then was sprayed onto rice seedlings. The sprayed rice seedlings were kept under moist and dark condition at 26 °C; For drought stress, PEG (polyethylene glycol) infused agar flasks were prepared by using 25% PEG (molecular weight 8000, Sigma, St. Louis) as described previously [44 (link)]. Then the 2-week-old seedlings were transferred into flask for further analyzing. The total RNA was extracted from roots of the seedlings exposed to the above stresses after 12 h for RNA analysis. For ABA treatment, seeds or 2-week-old seedlings were transferred into N6 solution with 0.5 or 1.0 μM cis, trans-ABA (Sigma Co.) respectively. For IAA treatment, the rice seedlings or seeds were transferred into N6 liquid medium containing 10 μM IAA (auxin/indole-3-acetic acid) for designated length of time.
6
Intracerebroventricular Drug Injection in Rodents
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In order to intracerebroventricular (i.c.v.) injection of drugs, the animals were anesthetized with intraperitoneal injection a mixture of ketamine and xylazine (90 and 10 mg/kg, respectively). Guide cannulas were implanted bilaterally at the coordinates of: 0.3 mm posterior to the bregma, ±1.0 mm lateral from the midline, and 2.5 mm depth to the cortical surface above the lateral ventricles (Paxinos et al., 1980 (link)). The implanted cannula was fixed to the skull surface by two screws and dental cement. After surgery, the animals were transferred to their home cages and given 1‐week recovery week before behavioral experiments. After recovery period, (±)‐cis, trans‐ABA (Sigma‐Aldrich, USA) was dissolved in the sterile saline solution (0.9% w/v sodium chloride) with DMSO at a ratio of 2:1 (v/v) and delivered into cerebral lateral ventricles for four consecutive days. A 27‐gauge needle connected by a polyethylene tube to a Hamilton syringe was used for the drugs injection (intracerebroventricularly, i.c.v.). The injection needle was remained in the place for additional 1 min to ensure a complete diffusion of the drugs.
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