Visualizing Autophagy in Arabidopsis Seedlings

Acridine Orange (AO; Invitrogen, Waltham, MA, USA) was used as a vital dye that penetrates cells. It stains nucleic acids and simultaneously, having pH-sensing properties, visualizes acidic intracellular structures including autophagosomes. The experiments were carried out on 10–12-day old Arabidopsis seedlings. The experimental groups of seedlings were incubated in phytotoxin solutions containing 100 mM sorbitol and 0.1% DMSO, supplemented with either 10 µg/mL STA or 50 µg/mL HBI. These concentrations of phytotoxins are known as non-lethal but inhibiting root growth [24 (link)]. After incubation in toxin-free control or in toxin-supplemented experimental solutions, the seedlings were transferred to an aqueous solution of 25 µM AO for 10 min, then washed for 1 min in a buffer of 100 mM sorbitol, 2 mM Tris, 1 mM MES, 0.1 mM KCl, 0.1 mM CaCl₂, pH 6.0 and analyzed under a confocal microscope LSM 780 (Carl Zeiss, Oberkochen, Germany) with a Plan-Apochromat objective 40×/1.4 Oil DIC. AO fluorescence was excited by a 488 nm argon laser; the fluorescence signal was detected in two channels, red and green (615–660 nm and 530–540 nm, respectively). To validate the method for assessing the pH of the apoplast using the AO fluorescent dye, experiments were performed in which the plasmalemma H⁺-ATPase inhibitor sodium orthovanadate (Na₃VO₄, Sigma-Aldrich) was used as a positive control.