α amanitin

α-amanitin (A2263, Sigma) was dissolved and diluted to 0.2 mg/ml in H₂O, and 1 nl of this solution was injected into embryos at the single-cell stage to deliver 0.2 ng of α-amanitin^{75 (link)}. Control embryos were injected with 1 nl of H₂O.

For the α-amanitin RNA sequencing data, A2780s SORBS2-knockdown or control cells were treated with 10 μg/mL α-amanitin (Sigma). Nine hours after α-amanitin treatment, RNA was isolated from the cells using a total RNA isolation kit and subsequently subjected to RNA sequencing. We compared and identified the different mRNAs in shControl and sh1-SORBS2 samples at 0 and 9 h after α-amanitin treatment. The differences between the sh1-SORBS2/shControl log fold changes were used as a measure of stability. For validation of the RNA sequencing results, relative transcript levels were assessed by qRT-PCR, and 18S was used as an endogenous normalization control. Statistical significance was determined using a one-tailed Student’s t-test. Cells were seeded at 2 × 10⁵ per well in six-well plates. Eighteen hours after seeding, DRB (Sigma) was added to the cells to a final concentration of 100 μM. RNA was isolated at 0, 2, 4, 6, and 8 h after DRB addition using a total RNA isolation kit with on-column DNase treatment (Norgen). Relative levels of the transcripts of interest were assessed by qRT-PCR, using 18S as the endogenous control. Half-life calculations were done using the formula t1/2 = ln 2/kdecay, where the decay constant was determined by plotting the data on a semilog scale and using nonlinear regression to find the best fit line (Graphpad Prism version 6).

Surface-sterilized Col-0 seeds were resuspended in 0,1% agarose and transferred to solid ½ MS medium supplemented with sterile filtered 4.6 ug/mL α-amanitin (Sigma-Aldrich, CAS 23109-05-9) and 9 ug/mL zebularine (Sigma-Aldrich; CAS 3690-10-6). For flagellin treatment, 2-week-old plants grown on ½ MS media supplemented with α-amanitin and zebularine were transferred to the equal ZA medium but also supplemented with 0.5 ug/mL flagellin (Flagellin from Salmonella typhimurium, Sigma-Aldrich, SRP8029-10UG). For ABA treatment, 2-week-old plants grown on ½ MS media supplemented with α-amanitin and zebularine were transferred to the equal ZA medium but also supplemented with abscisic acid at final concentrations of 100 uM. For heat-stress treatment, 2-week-old plants grown on ½ MS media supplemented with α-amanitin and zebularine were subjected to elevated temperature (4 °C for 24 h followed by 37 °C for 24 h, HS). For mild heat stress treatment 2 weeks-old Col-0 ddm1 plants were subjected to 30 °C for 24 h.