Quantifying Gibberellic Acid in Plant Tissues

To determine levels of GA in anthers and filaments, flowers at stage 13 (ref. 54 (link)) were dissected using a stereomicroscope. The GA levels in whole flowers at approximately stage 13 were also analysed. To measure the level of hormones in seedlings, 8-day-old plants grown on vertical plates were separated into shoots and roots. To collect developing seeds, siliques at 10 DAF were dissected using a stereomicroscope. All samples were frozen in liquid nitrogen and weighed after lyophilisation.
The samples were ground and homogenized in extract solution (Supplementary Table 2) with defined amounts of deuterium-labelled internal standards. The mixtures were incubated for 12 h at 4 °C and then centrifuged at 3,000g for 20 min at 4 °C. The supernatants were dried in a vacuum and dissolved in 1 ml of water containing 1% (v/v) acetic acid. After several steps of purification on solid phase columns, extracts were dried in a vacuum and dissolved in 20 μl of water containing 1% (v/v) acetic acid. The purification steps are summarized in Supplementary Tables 2 and 3. The LC–MS/MS system consisting of a quadrupole/time-of-flight tandem mass spectrometer (Triple TOF 5600, AB SCIEX), and a Nexera HPLC system (SHIMADZU) were used in these analyses. LC separations were performed at a flow rate of 400 μl min⁻¹ using the conditions presented in Supplementary Table 4. MS/MS conditions are presented in Supplementary Table 5. We used a software tool (MultiQuant 2.0, AB SCIEX) to calculate plant hormone concentrations from the LC–MS–MS data.