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Among these harvested plants, the plants t … Among these harvested plants, the plants that had fresh weight over 13 mg were used for GC-TOF/MS analysis (WT, n = 17; mto1, n = 13; and tt4, n = 20). For liquid chromatography-quadrupole-time-of-flight/mass spectrometry (LC-Q-TOF/MS) analysis, 3 biological replicates were prepared. All the plant materials were frozen immediately in liquid nitrogen to quench the enzymatic activity.<br /><br />
LC-Q-TOF/MS analysis<br />
Frozen leaves were homogenized in 5-μl extraction solvent (methanol/H2O [4:1 v/v]) per mg fresh weight of tissues by using a mixer mill at a frequency of 20 Hz for 3 min at 4°C. After centrifugation at 12,000 × g, the cell debris was discarded, and the extracts were centrifuged again. These supernatants were immediately used for flavonoid analysis. For flavonoid profiling, Waters Acquity UPLC™ system (Waters Co., Massachusetts, USA) fitted with a Q-ToF Premier mass spectrometer (Micromass MS Technologies, Manchester, UK) was used. Ultra-performance liquid chromatography (UPLC) was carried out on a UPLC™ BEH C18 column (100-mm length × 2.1-mm inner diameter, 1.7-μm particles, Waters Co.) at a flow rate of 0.5 ml/min at 35°C. The elution gradient comprised solvent A (0.1% trifluoroacetic acid in H2O) and solvent B (0.1% trifluoroacetic acid in acetonitrile) and the elution profile – 0 min, 100% A; 5 min, 11% B; 20 min, 13% B; 24 min, 100% B – using linear gradients in between the time points. A photodiode array (PDA) detector was used for the detection of UV-visible absorption in the range of 210–500 nm. The TOF mass analyzer was used for the detection of flavonoid glycosides [M+H]+ and fragment ions peak in a positive ion mode scan. The desolvation temperature was 450°C with a nitrogen gas flow rate of 600 l/h, capillary spray at 3.2 kV, source temperature at 150°C, and cone voltage at 35 V.<br />
The peaks in the plant extracts were identified based on retention times, UV visible absorption spectra, and mass fragmentation by tandem MS analysis as reported (Yonekura-Sakakibara et al. 2007). The amounts of each kaempferol glycoside and sinapoyl derivative were calculated using kaempferol (at 340 nm) or sinapic acid (at 340 nm) as a standard, respectively. d (at 340 nm) as a standard, respectively.
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