Abscisic acid aba

Plants were treated with indole-3-acetic acid (IAA) (PytoTechnology Lab., Lenexa, USA) and abscisic acid (ABA) (Sigma-Aldrich, Sidney, Australia). Hormone stock solutions were made with 100% ethanol. Wheat tillers were collected and dipped in hormone solutions for 9 h containing either 100 μM IAA or 100 μM ABA, to a final concentration of 0.05% ethanol. A 0.05% ethanol solution was used as a control treatment. For the drought treatment, plants were well watered until the stage of flag leaf emergence and water withheld until wilting. After sample collection, plants were re-watered. The effects of the cyclic drought treatment was assessed from the percentage of fertility of three spikes from well-watered and treated plants calculated according to Tucker et al. (2017) [16 (link)].
GUS activity in anthers from treated transgenic lines was analyzed by histochemical staining using X-gluc as previously described. Anthers containing developing microspores were staged by acetocarmine staining. Six spikes were used for each treatment.

Deionised water was filtered through Direct-Q UV (Millipore), methanol and isopropanol were purchased from Fisher Chemicals, NaOH from Agilent Technologies, acetic acid and formic acid from Sigma Aldrich. Standards were used to develop the UHPLC and MS/MS methods: abscisic acid (ABA, Sigma), benzoic acid (BA, Fluka), brassinolide (BR, OlChemIm), cis-12-oxo-phytodienoic acid (cis-OPDA, OlChemIm), castasterone (CS, OlChemIm), cathasterone (CT, OlChemIm), gibberellins A₁, A₄ and A₇ (GA₁, GA₄, GA₇ OlChemIm), gibberellic acid (GA₃, Fluka), indole-3-acetic acid (IAA, Serva), indole-3-butyric acid potassium salt (IBA, Sigma), jasmonic acid (JA, OlChemIm), jasmonoyl isoleucine (JA ILE, OlchemIm), 6-furfurylaminopurine (kinetin, KIN, Duchefa), orobanchol (Oro, OlChemIm), salicylic acid (SA, Sigma), trans zeatin (t-zea, Sigma), 6-(γ,γ-dimethylallylamino)purine (2iP, Duchefa). ²H₃ CS and ²H₆ ABA were purchased from OlChemIm and used as internal standards spiked before extraction. Standards of 2-heptanone, 4-heptanone, pentanamide, furfural and azelaic acid were purchased from Sigma-Aldrich to confirm the identification of putative metabolites of interest obtained from the non-targeted analysis by UHPLC-QTOF-MS/MS.

Abscisic acid (ABA) (Sigma Aldrich, Poznan, Poland) was added to the modified Gamborg medium on day of inoculation or 25 day of culture when the P. quinquefolium hairy root culture was in its stationary growth phase. ABA was prepared as a stock solution, sterilized through a Millipore filter of pore size 0.20 μm (Merck Millipore Ltd., Burlington, MA, USA), and then added to the liquid media at final concentrations of 0 (control), 0.1, 0.25, 0.5, and 1 mg·L⁻¹. The effect on the expression of the HMGR gene was examined after 6, 12, and 24 h of elicitor treatment. Biomass and ginsenoside accumulation in hairy root cultures of P. quinquefolium were measured after 1, 3, and 28 days of abscisic acid treatment.

Contents of endogenous ABA and IAA were analyzed in the in vitro plantlets grown at 0 and 4% PEG. Shoots with leaves were taken from 1-week-old plantlets and divided into two groups. One group was used for analysis of ABA and IAA, and the other for measurement of transcription level of ABA and IAA biosynthetic genes, as described below. Samples were frozen in liquid nitrogen and stored at −80°C until usage. Abscisic acid (ABA) and indoleactic acid (IAA) were provided by Sigma (St. Louis, MO, USA). At the beginning of extraction, 1000 Bq of each ABA and IAA, accordingly, were added to monitor the losses during purification. The extraction and purification of ABA and IAA were conducted, according to by Dobrev et al. (2005 (link)). The purified extract solution was transferred into 2-ml centrifuge tubes containing 0.3 g polyvinylpolypyrolidone (PVPP) and then kept at −20°C for the measurements of ABA and IAA in water breeze HPLC system (Waters 2489 UV/Visible Detector), using wavelength at 254 nm, velocity at 0.7 ml min⁻¹ and sample quantity of 10 μm and column temperature at 30°C. The dried fraction containing ABA and IAA were, respectively, injected into the HPLC system for measurements, as described by Li et al. (2013 (link)).

Fresh seeds of WT Arabidopsis thaliana ecotype Columbia (Col-0), transgenic lines expressing PHY-US417^{51 (link)} and the ipk1-1 mutant^{35 (link)} were collected from plants grown side by side under the same standard growth conditions. Seeds were surface sterilized with 70% ethanol for 10 min, rinsed with water three times and plated onto Murashige and Skoog (MS) media^{78 (link)} without or with 125 mM NaCl (Sigma), 150 mM mannitol (Sigma) or increasing concentrations (1, 2.5 and 5 µM) of abscisic acid ABA (Sigma). Seedlings were grown for two weeks on vertical plates under light/dark cycle conditions of 16/8 h, light intensity of 250 µmol m⁻² s⁻¹, and temperature of 22–24 °C in growth chamber. For growth assessment under osmotic stress treatments, one-week-old seedlings were transferred onto MS agar media only or containing 300 mM mannitol for 2 additional weeks. Plates were grown as aforementioned. After photo documentation, root lengths were measured using the OPTIMAS software (OPTIMAS Image Analysis system 6.1, Media Cybernetic). Then, shoots were collected for measurements of AsA, chlorophyll, H₂O₂ and MDA levels as well as antioxidant enzymatic activities (see below).

Plant hormones and their synthetic analogs used in the experiments were purchased from Sigma. They were abscisic acid (ABA, Sigma A1049), indole-3-acidic acid (IAA, Sigma I2886), indole-3-butyric acid (IBA, Sigma I5386), 1-naphthalene-acetic acid (NAA, Sigma N0640), 2,4-dichlorophenoxy acetic acid (2,4-D, Sigma D6679) and 3,6-dichloro-2-methoxybenzoic acid (Dicamba or DIC, Sigma D5417). Plant hormones were dissolved in 10 volumes of ethanol according to manufacturer’s instructions and then diluted with water to the desired concentrations before uses. The control received the same amount of ethanol equal to those in dissolving the chemicals.

Seeds of Cherry belle with uniform size and plumpness were put into a 9 cm diameter petri dish lined with three pieces of filter paper and wetted with 4 ml deionized water. Three days after imbibition, the seeds were treated with deionized water or solutions including 100 μM abscisic acid (ABA, Sigma A1049), 100 μM 6-Benzylaminopurine (6-BA, Sigma B3274), 100 μM Gibberellic acid (GA₃, Sigma G7645), 50 μM alpha-Naphthaleneacetic acid (NAA, Sigma N0640), and 50 μM Ethephon (Sigma 45473). At least 300 seeds were used for each treatment. Root samples were collected 12 and 24 h after treatment, immediately frozen in liquid nitrogen, and stored at −80 °C for further use.