Transsonic 460

Pollen material from wild-type and transgenic Arabidopsis lines were collected as described above by the vacuum-cleaner method (Johnson-Brousseau and McCormick, 2004) . Ten milligrams of freshly harvested pollen was degreased with 500 mL of hexane. The suspension was centrifuged at 4°C, 15 min, 20,817g. The hexane supernatant was removed and used for additional GC-MS analysis (Supplemental Figure 3). The pellet was completely dried at 30°C for 10 min to evaporate the remaining hexane and resolved in 200 mL of 90% (v/v) methanol. The samples were incubated for 15 min in a sonication bath (Transsonic 460; Elma) at 35 kHz. Afterward, the samples were centrifuged for 15 min at 20,817g at 4°C to separate the residual pollen material. These methanolic extracts were further directly used for liquid chromatography-mass spectroscopy (LC-MS) and HPTLC analysis.

Dexamethasone was quantified by high performance liquid chromatography (HPLC) using a liquid chromatograph with a pump M520, a UV detector M490E, an autosampler 712D WTSP and the Empower Login HPLC System Manager Software, all by Waters (MA, USA). The chromatographic separation was achieved with a C18 column Tracer Excel 120 ODSA (particle size 5 µm, 150 mm x 4mm; Teknokroma, Barcelona, Spain). The mobile phase flow was set at 1 mL/min and the injection volume was 20 µL. The absorbance of the eluent was monitored at 254 nm. All the analyses were performed at 45 ± 0.5ºC.
The composition of the mobile phase A was methanol:ACN:water (3:3:4). The mobile phase B was composed of 100% ACN. Both A and B were vacuum-filtered through 0.45 µm nylon millipore membrane (Merck, Darmstadt, Germany), and degassed by ultrasonication for 15 minutes before use (Elma Transsonic 460, Singen, Germany). A gradient elution method was employed and the chromatograph was programmed as follows: 100% A for 10 min, followed by 0 to 100% B over 15 min, then 100 to 0% B over 5 min and finally 100% A over 5 min. The HPLC method was validated with respect to linearity, accuracy and reliability in the range of concentrations of 2-20 µg/mL.

Hydrogenation under atmospheric hydrogen pressure in an ultrasonic bath Alkynes (Alfa Aesar, 98%) were hydrogenated over the prepared Pd-catalysts at room temperature (RT, 25±1 °C) and under atmospheric hydrogen pressure (AHP, H2 balloon). 5 mg of Pd-catalyst was added to either 50 mL of hexane (Sigma-Aldrich, ≧99%), or ethanol (Fluka, 96%), containing 0.58 mmol of the alkyne. The reactor was evacuated to 0.02 MPa using a vacuum pump and then flushed with nitrogen (Sapio, grade 6.0), before the reaction. The reactor was then evacuated to 0.02 MPa once more and filled with hydrogen (Sapio, grade 4.5) via a connected H2 balloon. The reaction was performed in an ultrasonic bath (Elma, Transsonic 460, 35 kHz, 85 W) filled with 1.5 L water while liquid temperature was controlled by adding ice. Aliquots (100 µl) of the solution were periodically extracted from the reaction system using an airtight syringe. These were then diluted with 900 µl cyclohexane (Alfa Aesar, 99.9%) and analyzed using GC/MS (Agilent Technologies 6850 Network GC system equipped with a 5973 Network Mass Selective Detector and an HP-5MS capillary column).
The same reaction media were placed in a water bath and the reaction was performed without sonication under stirring at 500 rpm. This was done in order to provide a basis for valid comparison.