Gibberellic acid

For H₂O₂ treatment, H₂O₂ was freshly prepared as 1 M stock solution with sterile double stilled water. For protoplast treatments with different concentrations of H₂O₂, H₂O₂ was further prepared as 100× stock with protoplast buffer (W5 solution). Protoplast cells were then treated with different concentrations of H₂O₂ for 24 h. For each treatment, at least three biological replicates were conducted. After 24 h treatment, samples were stained with Fluorescein Diacetate (FDA) (Cat. #F1303, Invitrogen) for viability observation. For 50 µL of sample, 2 µL of FDA was added. Immediately after staining, the samples were observed under Olympus BX53 Epi-fluorescence microscope with green channel. The ratio of green cells (living cells) to total cells was calculated as viability rate. All steps and chemical treatments were performed at room temperature.
Protoplast cells co-treated with H₂O₂, antioxidants or Gibberellic acid (Alfa Aesar) were conducted as described above. Uric acid (Cat. # A13346, Thermo-Fisher Scientific) was dissolved in protoplast buffer (W5 solution). Rutin hydrate (Cat. # R5143, Sigma-Aldrich) was dissolved in DMSO as stock, Gibberellic acid (Cat. #A17843, Alfa Aesar) was dissolved in ethanol as stock.