Figure 10.

KIN10 Is Required for the Autophagy Induced by Prolonged Fixed-Carbon Starvation in the atg11 Mutant.

(A) Immunoblot detection of the free GFP released during the autophagic degradation of GFP-ATG8a reporter during fixed-carbon starvation. One-week-old atg11-1, KIN10-OE atg11-1, and KIN10-RNAi atg11-1 seedlings expressing the GFP-ATG8a reporter were grown on MS solid medium with 1% (w/v) Suc and then transferred to sucrose-deficient liquid medium and darkness for the indicated times. Immunoblot analysis of total seedling extracts was performed as in Figure 1A. The GFP-ATG8a fusion and free GFP are indicated by closed and open arrowheads, respectively. Immunoblot detection of histone H3 was used to confirm nearly equal protein loading.

(B) Quantification of the free GFP:GFP fusion ratio of the GFP-ATG8a reporter used in (A). Levels of free GFP and the GFP fusion were determined by densitometric scans of the immunoblots. Each data point represents the mean ± sd of three independent biological replicates.

(C) to (E) Expression levels of KIN10 and ATG6 affect the formation of GFP-ATG8-positive puncta in the atg11-1 mutant. In (C), protoplasts were prepared from atg11-1, KIN10-OE atg11-1, and KIN10-RNAi atg11-1 plants expressing GFP-ATG8a. In (D), ATG6-HA or an empty vector control was transiently expressed in protoplasts prepared from atg11-1 and KIN10-RNAi atg11-1 plants. Protoplasts were then transferred to MS liquid medium without Suc (−C) in darkness for 24 h before confocal fluorescence microscopic observation. (E) represents the numbers of puncta per section in the protoplasts shown in (C) and (D). The data represent average values calculated from three independent experiments. Letters indicate values that are statistically different from one another (P < 0.05) as determined using one-way ANOVA followed by multiple-comparisons Tukey’s test; n = 90 protoplasts per treatment. UN, untreated protoplasts.