Measurement of strigolactone content
Root samples (1 g fresh weight) were ground into powder in liquid nitrogen and extracted in 10 mL pre-cooling acetone at 4oC overnight. After centrifuged (12000 g , 4 oC, 5 min), the supernatant was blow-dried with nitrogen and dissolved in 2 mL 20% acetone. The mixture was subjected to a Sephadex HLB column and eluted by 2 mL acetone. The eluate was blow-dried with nitrogen and dissolved in 200 μL acetonitrile. 2 μL mixture was subjected to a poroshell 120 SB-C18 reverse-phase column (2.1×150 mm, 2.7 μm, Agilent, USA) using acetonitrile:H2O as a mobile phase at a flow rate of 0.3 mL/min and the column temperature was set at 30oC. The gradient parameter of HPLC (Aglient1290, Aglient, USA) was performed as follow: (1) acetonitrile:H2O (4:1) for 30 sec; (2) from acetonitrile:H2O (4:1) to acetonitrile:H2O (1:9) for 150 sec; (3) acetonitrile:H2O (1:9) for 120 sec; (4) from acetonitrile:H2O (1:9) to acetonitrile:H2O (4:1) for 6 sec; (5) acetonitrile:H2O (4:1) for 173 sec. The column effluents were subjected to Qtrap6500 mass spectrometer (Applied Biosystems, USA) in the ESI and MRM mode. The peaks showing activity for 5-deoxystrigol (5-DS) and strigol were detected at 4.04 min. The selected reaction monitoring conditions for protonated or deprotonated SLs were showed in Supplementary Table S1.
In the present study, we used external standard method to detect the contents of 5-DS and strigol in our samples. 5-DS and strigol were supplied by StrigolLab (StrigolLab, Italy) and dissolved in acetonitrile. Different concentration (0.5 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL and 40 ng/mL) of 5-DS and strigol were prepared for standard curves. The R2 value of 5-DS and strigol were 0.99063 and 0.99797, respectively.