Figure 1.
Generation of RNAi-AtTOR transgenic lines.
A Total RNA was extracted from 7-day-old WT and five independent RNAi-AtTOR lines, RNAi-1 to RNAi-5, followed by RT-PCR analysis. 18S was used as a loading control. B The root length of 10-day-old WT, RNAi-2, RNAi-3 seedlings was measured on both MS medium and MS medium lacking nitrogen (MS-N). C The fresh weight of WT, RNAi-2, RNAi-3 seedlings grown on MS medium was measured at the stated times. *** indicates P<0.001; ** indicates P<0.01 and * indicates P<0.05. Error bars indicate standard error.
Figure 2.
RNAi-AtTOR plants have constitutive autophagy.
A Seven-day-old WT and RNAi-1 to RNAi-5 seedlings were stained with MDC and observed by fluorescence microscopy. Arrows indicate MDC-stained autophagosomes. B GFP-AtATG8e-labeled autophagosomes were visualized by fluorescence microscopy in 7-day-old GFP-AtATG8e and RNAi-2×GFP-AtATG8e seedlings. Arrows indicate GFP-labeled autophagosomes. Insets show an enlargement of the boxed areas. Scale bar = 50 µm for main figures, 10 µm for insets. C Seven-day-old GFP-AtATG8e and RNAi-2× GFP-AtATG8e seedlings were transferred to liquid MS medium containing 1 µM concanamycin A (+concA) or DMSO (-concA) as a solvent control for 12 h, followed by both fluorescence and DIC microscopy. Scale bar = 50 µm.
Figure 3.
Some ATG genes are up-regulated in RNAi-AtTOR plants.
A RNA was extracted from 8 mm of the root tips from seven-day-old WT and RNAi-2 lines, followed by RT-PCR analysis of the indicated genes. 18S was used as a loading control. B Densitometry was used to quantify the relative amounts of RT-PCR product from at least three independent replicates, with the wild-type control value set as 1. ** indicates P<0.01 and * indicates P<0.05. Error bars indicate standard error. C 7-day-old AtATG18apro: GUS and RNAi-2×AtATG18apro:GUS seedlings were collected and submerged in GUS staining solution for 16 h, destained in 70% ethanol for 16 h and observed with a light microscope. Scale bar = 1 mm. D 7-day-old AtATG18apro: GUS and RNAi-2×AtATG18apro:GUS root tissue extracts were added to 1 mM MUG solution to assay GUS activity. At 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 16 h, the reactions were terminated with 0.2 M Na2CO3 followed by measurement of MU fluorescence. Data was collected from 3 independent replicates, with AtATG18apro: GUS 0 h value set as 1. ** indicates P<0.01 and * indicates P<0.05.
Figure 4.
AtTOR-regulated autophagy is dependent on AtATG18a.
A MDC staining was performed on seven-day-old RNAi-2 and RNAi-2×RNAi-AtATG18a seedlings and observed by fluorescence microscopy. The white arrow indicates an MDC-stained autophagosome. B Seven-day-old RNAi-2 and RNAi-2×RNAi-AtATG18a seedlings were transferred to liquid MS medium containing 1 µM concanamycin A (+concA) or DMSO (-concA) for 12 h, followed by DIC microscopy. Scale bar = 50 µm.
Figure 5.
NADPH oxidase inhibitor does not inhibit autophagy in RNAi-AtTOR plants.
A 7-day-old WT and RNAi-2 seedlings were transferred to liquid MS medium plus or minus 20 mM imidazole for 4 h, or WT seedlings were transferred to liquid MS medium plus 0.16 M NaCl plus or minus 20 mM imidazole for 4 h. Autophagosomes were detected by MDC staining. Arrows indicated MDC-stained autophagosomes. B 7-day-old GFP-AtATG8e and RNAi-2× GFP-AtATG8e seedlings were transferred to liquid MS medium plus or minus 20 mM imidazole for 4 h, or GFP-AtATG8e seedlings were transferred to liquid MS medium plus 0.16 M NaCl plus or minus 20 mM imidazole for 4 h. Autophagosomes were detected by fluorescence microscopy via a FITC-specific filter. Arrows indicated GFP-labeled autophagosomes. Scale bar = 50 µm. C Seven-day-old RNAi-2× GFP-AtATG8e seedlings were transferred to liquid MS medium containing 1 µM concanamycin A (+concA) or DMSO (-concA) as a solvent control for 12 h, or liquid MS medium plus 20 mM imidazole containing 1 µM conc A or DMSO for 12 h followed by both fluorescence microscopy and DIC microscopy. Scale bar = 50 µm.
Table 1.
Primers used for generating the RNAi-AtTOR construct and for RT-PCR analysis of AtTOR, AtATG18a, AtATG9 and AtATG8s.