Inhibition of SnRK1 activity by T6P inhibits autophagy under abiotic stress.

Seven-day-old GFP-ATG8e seedlings were transferred to ½ MS liquid medium supplemented with 0.1 mM T6P for 3 hours as control, or liquid medium supplemented with 160 mM NaCl for 6 hours and 0.1 mM T6P for the last 3 hours of treatment (A), liquid medium supplemented with 350 mM mannitol for 6 hours and 0.1 mM T6P for the last 3 hours of treatment (B), ½ MS plates lacking sucrose for 4 days in the dark followed by 0.1 mM T6P treatment in liquid medium for 3 hours (C), ½ MS plates lacking nitrogen for 4 days followed by 0.1 mM T6P treatment in liquid medium for 3 hours (D), liquid medium supplemented with 0.1 mM T6P for 3 hours and 10 mM hydrogen peroxide added for the last 2 hours (E), or liquid medium supplemented with 2 mM DTT for 6 hours and 0.1 mM T6P for the last 3 hours of treatment (F). Autophagosomes were imaged using epifluorescence microscopy and counted. Addition of T6P blocked the activation of autophagy in most conditions. In osmotic stress, autophagy was reduced but not completely blocked by T6P. Different letters denote statistical significance for three biological replicates with at least 10 frames per replicate, p<0.05, t-test. Error bars indicate standard error. (G) Confocal images of roots of GFP-ATG8e-expressing seedlings under control conditions, ER stress and salt stress as representative stresses. The insets show enlargements of the indicated boxes. White arrows point to autophagosomes. Scale bars = 20 μm.