Material and methods


GC-MS analyses of root metabolites were carried out as previously described (Broeckling et al., 2005). Root hairs and stripped roots were lyophilized until dry. The dried root tissues were homogenized using mortar and pestle, and 6 mg were transferred into a 4 ml glass vial. Metabolites were extracted by adding 1 ml of chloroform containing 10 µg/ml of docosanol (the standard for non-polar metabolites), vortexed and incubated for 45 min. at 50ºC. HPLC-grade water (1.5 ml), containing 25 µg/ml of ribitol (the standard for polar metabolites), was added and incubated a second time at 50ºC for 45 min. The two phases were separated by centrifugation at 2900 g for 30 min at 4ºC. One ml of each layer was collected into a vial and dried in a rotary evaporator for the polar layer or under nitrogen for the non-polar layer. Methoxyamine hydrochloride (50 µl at 15mg/ml) in pyridine was added to the polar metabolites. Compounds were sonicated until they were completely dissolved, and incubated at 50ºC for at least 1 h for methoximation. Silylation was performed by incubating the samples 1h at 50ºC with 50 µl MSTFA and 1% TMCS (Pierce Biotechnology, Rockford, IL, USA). The sample was equilibrated to room temperature, transferred to a 200 µl glass insert, and analyzed using an Agilent 6890 GC coupled to a 5973 MSD scanning from m/z 50–650. One µl of the sample solution was injected at a 15:1 split ratio, and the inlet and transfer line were held at 280 ºC. Separation was achieved on a 60 m DB-5MS column (J&W Scientific, 0.25 mm ID, 0.25 lm film thickness) at a constant flow of 1.0 ml/min with a temperature program of 80 ºC for 2 min, then ramped at 5 ºC/min to 315 ºC and held for 12 min.

The non-polar compounds were re-suspended in 0.8 ml of chloroform and hydrolyzed for 4 h at 50ºC by adding 0.5 ml of HCl (1.25 M) in methanol. HCl and solvent were removed by evaporation under nitrogen. Solubilization was achieved by sonication in 70 µl of pyridine and incubation at 50ºC. Derivatization was performed, as described above, by using 30 µl MSTFA and 1% TMCS. Metabolites were analyzed by GC-MS as described above for the polar compounds using a 1:1 split ratio.


Root hairs and stripped roots were lyophilized until dry and ground to a fine powder. Ten mg of the powder was extracted for 2h with 1 ml of 80% methanol containing 0.018 mg/ml of umbelliferone as an internal standard. Samples were centrifuged at 2900g for 30 min at 4 ºC and the supernatants were collected. Five μl of the supernatant was injected into Waters ultra performance liquid chromatography (UPLC) system coupled to a quadrupole-time of flight mass spectrometer (QTOF Premier). Separation of metabolites was achieved on a 2.1 × 150 mm i.d., 1.7 μm UPLC BEH C18 column (Waters Acquity) using the following gradient: mobile phases B (acetonitrile) increased from 5% to 70% over 30 min, then to 95% in 3 min, held at 95% for 3 min and returned to 95% mobile phase A (0.1% acetic acid in water) for equilibration for 3 min. The flow rate of the mobile phases was 0.56 ml/min and the column temperature and the autosampler temperature were maintained at 60°C and 4°C respectively. Mass spectral data were acquired from m/z 50 to 2000 in the negative ion electrospray mode, with the nebulization gas at 850 l/h (350°C) and the cone gas at 50 l/h (120°C). Raffinose (m/z 503.1612) was used as the reference compound in the independent lock-mass mode with the lock mass scan (1s) collected every 10 s for the accurate mass measurement. The concentration of raffinose was 50 fmol/mL and the flow rate 0.2 mL/h.