The endogenous ABA content was quantified from the frozen samples by following the protocols of Qi et al.
[66 (link)] and Kamboj et al.
[67 ]. Aerial parts of the plant samples were extracted with 30 ml of extraction solution containing 95% isopropanol, 5% glacial acetic acid, and 20 ng of [(±)–3,5,5,7,7,7–d6]–ABA. The filtrate was concentrated by a rotary evaporator. The residue was dissolved in 4 ml of 1 N sodium hydroxide solution, and then washed three times with 3 ml of methylene chloride to remove lipophilic materials. The aqueous phase, brought to approximately a pH of 3.5 with 6 N hydrochloric acid was partitioned three times into ethyl acetate (EtOAc). EtOAc extracts were then combined and evaporated. The dried residue was dissolved in phosphate buffer (pH 8.0) and then run through a polyvinylpolypyrrolidone (PVPP) column. The phosphate buffer was adjusted to pH 3.5 with 6 N HCl and partitioned three times into EtOAc. EtOAc extracts were combined again and evaporated. The residue was dissolved in dichloromethane (CH2Cl2), and passed through a silica cartridge (Sep-Pak; Water Associates, Milford, Massachusetts, USA) pre-washed with 10 ml of diethyl ether: methanol (3:2, v/v) and 10 ml of dichloromethane. ABA was recovered from the cartridge by elution with 10 ml of diethyl ether (CH3-CH2)2O: methanol (MeOH) (3:2, v/v). The extracts were dried and methylated by adding diazomethane for GC/MS-SIM (6890 N network GC system, and the 5973 network mass-selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuset, Manchester, UK) data system software was used to monitor responses to ions with an m/e of 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA.
[66 (link)] and Kamboj et al.
[67 ]. Aerial parts of the plant samples were extracted with 30 ml of extraction solution containing 95% isopropanol, 5% glacial acetic acid, and 20 ng of [(±)–3,5,5,7,7,7–d6]–ABA. The filtrate was concentrated by a rotary evaporator. The residue was dissolved in 4 ml of 1 N sodium hydroxide solution, and then washed three times with 3 ml of methylene chloride to remove lipophilic materials. The aqueous phase, brought to approximately a pH of 3.5 with 6 N hydrochloric acid was partitioned three times into ethyl acetate (EtOAc). EtOAc extracts were then combined and evaporated. The dried residue was dissolved in phosphate buffer (pH 8.0) and then run through a polyvinylpolypyrrolidone (PVPP) column. The phosphate buffer was adjusted to pH 3.5 with 6 N HCl and partitioned three times into EtOAc. EtOAc extracts were combined again and evaporated. The residue was dissolved in dichloromethane (CH2Cl2), and passed through a silica cartridge (Sep-Pak; Water Associates, Milford, Massachusetts, USA) pre-washed with 10 ml of diethyl ether: methanol (3:2, v/v) and 10 ml of dichloromethane. ABA was recovered from the cartridge by elution with 10 ml of diethyl ether (CH3-CH2)2O: methanol (MeOH) (3:2, v/v). The extracts were dried and methylated by adding diazomethane for GC/MS-SIM (6890 N network GC system, and the 5973 network mass-selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuset, Manchester, UK) data system software was used to monitor responses to ions with an m/e of 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA.
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