Sclareol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Campesterol, stigmasterol, and lovastatin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Sclareol, Campesterol, stigmasterol, and lovastatin were dissolved in methanol and diluted in water to various concentrations. Methanol concentrations did not exceed 0.1% (v/v) in any experiment.
The procedure of chemical treatment, including fractions obtained from fractionation, was performed according to previous reports [16 (link),18 (link)]. Briefly, plants were removed from the fertilizer, transferred to a glass Petri dish (9 cm in diameter) containing sterile distilled H2O, and preincubated for 24 h to diminish the influence of any stress wounds caused by the transfer. After discarding water with a pipette, plant roots were treated by gently adding a solution containing adequate concentrations of each fraction or compound for 48 h. The treated plants were harvested and used for chlorophyll measurements, gene expression analysis, and phytosterol quantification. Five to six plants were used for each treatment and regarded as one biological replicate.
For the lovastatin treatment, we used 100 μM lovastatin, 100 μM Sclareol, and a mixture of 100 μM lovastatin and 100 μM Sclareol in a 50:50 ratio.
The procedure of chemical treatment, including fractions obtained from fractionation, was performed according to previous reports [16 (link),18 (link)]. Briefly, plants were removed from the fertilizer, transferred to a glass Petri dish (9 cm in diameter) containing sterile distilled H2O, and preincubated for 24 h to diminish the influence of any stress wounds caused by the transfer. After discarding water with a pipette, plant roots were treated by gently adding a solution containing adequate concentrations of each fraction or compound for 48 h. The treated plants were harvested and used for chlorophyll measurements, gene expression analysis, and phytosterol quantification. Five to six plants were used for each treatment and regarded as one biological replicate.
For the lovastatin treatment, we used 100 μM lovastatin, 100 μM Sclareol, and a mixture of 100 μM lovastatin and 100 μM Sclareol in a 50:50 ratio.