Ultra performance liquid chromatography (uplc)

Frozen plant tissues were ground to a uniform powder and extracted in 80% methanol. The dried samples were dissolved in 80% methanol and analysed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI/MS) using a Waters UPLC connected to a Bruker micrOTOF-Q mass spectrometer. The analyses were performed in a gradient mobile phase consisting of 0.5% formic acid (v/v) in water (A) and 0.5% formic acid (v/v) in acetonitrile (B). The m/z range of the recorded spectra was 50–1000. The analyses were performed in the ion-positive mode for phenolics and ion-negative mode for carboxylic acids. For details, see Staszków et al., 2011 (link).

Four‐day‐old suspension cell cultures (overexpressing MtABCG20 or transformed with EV) were filtered, washed, and suspended in fresh, ice‐cold growth medium. After the addition of ABA (250 μm) as a substrate, the cells were incubated for 30 min at 4°C with agitation (60 rpm). After incubation, the cells were filtered, washed and transferred to fresh, growth medium (T0), and then incubated with agitation (60 rpm) at 22°C/18°C. Samples (5 ml of cell culture) were collected at the defined time points, filtered and frozen. Frozen cells were ground at 4°C with mortar and pestle, and extracted with 3 ml of 80% methanol. Dried extracted samples were dissolved in 200 μl of 80% methanol and analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI/MS) using a Waters UPLC connected to a Bruker micrOTOF‐Q mass spectrometer (Staszkow et al., 2011). Deuterated ABA was used as the internal standard.

Frozen root tissue (500 mg) was grounded and extracted with 80% methanol. Root exudates were extracted from the medium by SPE (Solid Phase Extraction) method using octadecylosilane matrix and methanol according to Staszków et al. (2011) (link). Luteolin was used as an internal standard. Samples were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI/MS) using a Waters UPLC coupled with Bruker micrOTOF-Q mass spectrometer. The analysis was performed in a gradient mobile phase consisting of 0.5% formic acid (v/v) in water (A) and 0.5% formic acid (v/v) in acetonitrile (B). The m/z range of the recorded spectra was 50–1,000. Analyses were performed in the ion-positive mode.