Fig. 3: Artificially truncated ATG8 restores autophagic activity in the ATG8 loss-of-function mutant.

A CLSM of Arabidopsis roots expressing GFP–ATG8FΔC in WT and atg8a-i backgrounds treated with AZD/ConA. White arrowheads indicate autophagic bodies. Scale bars, 20 μm. B Vacuolar puncta quantification for the samples (n = 12 biological replicates) illustrated in (A). C Western blot detection of GFP–ATG8FΔC cleavage in WT and atg8a-i backgrounds shows similar levels of free GFP accumulation in both WT and atg8a-i backgrounds upon induction of autophagy, suggesting that the introduction of GFP–ATG8FΔC restored autophagic flux in the ATG8 loss-of-function mutant. The numbers under corresponding lanes represent the integrated density of the free GFP band expressed as % of the total signal intensity for the corresponding sample. PS Ponceau S, –Ctrl, protein extract from Arabidopsis seedlings not expressing GFP-tagged fusions. D Root phenotype of 11-day-old seedlings incubated for 6 days on control or nitrogen-depleted medium. Expression of the artificially truncated ATG8FΔC isoform is sufficient to alleviate the autophagy-deficient phenotype of atg8a-i. Seedlings were grown for 5 days on standard 0.5× MS medium, checked for GFP signal, and then transferred to plates with standard 0.5× MS (Control) or nitrogen-depleted (–N) medium to be imaged for 7 days. Scale bar, 1 cm. E Quantification of root growth on control and –N medium (n = 288 seedlings per treatment) in the WT and atg8a-i expressing the artificially truncated GFP–ATG8FΔC (illustrated in D).