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. 2019 Mar 31;15(12):2126–2141. doi: 10.1080/15548627.2019.1596496

Figure 3.

Figure 3.

Overexpression of PRF3-MYC does not affect nocturnal or salt stress-induced autophagy. (a) Representative confocal images of CFP-ATG8f-labeled autophagic structures in cLUC-MYC- or PRF3-MYC-expressing leaves with (lower panel) or without (upper panel) E-64d during nocturnal autophagy. CFP-ATG8f was co-expressed with cLUC-MYC (control) or PRF3-MYC in leaves for 2 days before imaging at the time point D4. 100 μM E-64d was infiltrated into leaves for 12 h to block vacuolar degradation. Scale bars: 10 μm. (b) Relative autophagic activity in PRF3-MYC-expressing leaves (with or without E-64d) during nocturnal autophagy upregulation indicated by CFP-ATG8f. The autophagic activity in cLUC-MYC-expressing leaves without E-64d was set to 1.0. (c) GFP-ATG8 processing assay in control and PRF3-MYC-expressing leaves with or without E-64d during nocturnal autophagy. ProATG8f::GFP-ATG8f was coexpressed with cLUC-MYC or PRF3-MYC for 48 h. Open and closed arrowheads indicate the GFP band and GFP-ATG8f band respectively. RBCL stained by Ponceau indicates equal loading (lower panel). (d) Representative images of CFP-ATG8f-labeled autophagic structures in cLUC-MYC- or PRF3-MYC-expressing leaves with (lower panel) or without (upper panel) E-64d during salt stress-induced autophagy. CFP-ATG8f was co-expressed with cLUC-MYC (control) or PRF3-MYC for 2 days before imaging.150 mM NaCl was infiltrated into leaves for 1 h to induce autophagy. 100 μM E-64d was infiltrated into leaves for 12 h. Scale bars: 10 μm. (e) Relative autophagic activity in PRF3-MYC-expressing leaves (with or without E-64d) during salt stress-induced autophagy. Autophagic activity in cLUC-MYC-expressing leaves without E-64d was set to 1.0. More than 200 cells were quantified in each treatment. Values are means ± SE from 3 independent experiments. Different letters indicate significant difference (ANOVA, p < 0.05). (f) GFP-ATG8 processing assay in control and PRF3-MYC-expressing leaves with or without E-64d during salt stress-induced autophagy. Open and closed arrowheads indicate the GFP band and GFP-ATG8f band respectively. RBCL stained by Ponceau indicate equal loading (lower panel).