(A) Measurement of proteaphagy upon nitrogen and/or fixed-carbon starvation in 5 day-old Arabidopsis seedlings by monitoring the release of free GFP from the CP and RP subunits PAG1-GFP or RPN5a-GFP, respectively. PAG1:PAG1-GFP pag1-1 and RPN5a:RPN5a-GFP rpn5a-2 seedlings were switched from growth in the light on nutrient-rich (+N +C) medium to either growth in the light on medium lacking nitrogen (–N), or growth in the dark on media lacking either carbon alone (–C) or both nitrogen and carbon (–N –C). Total protein extracts prepared from seedlings harvested at the indicated times were assayed for GFP release by immunoblot analysis with anti-GFP antibodies. Open and closed arrowheads locate the GFP fusion and free GFP, respectively. Immunodetection of histone H3 was used to confirm near equal protein loading. Rates of bulk autophagy were measured by the release of GFP from GFP-ATG8a in the same manner as above (right panel). (B) Quantification of the free GFP/GFP fusion ratios of the PAG1-GFP, RPN5a-GFP and GFP-ATG8a reporters upon switching from +N +C medium to –N, –C, or –N –C media. Levels of the GFP fusion and free GFP were determined by densitometric scans of the immunoblots shown in panel (A). Each data point represents the mean (±SD) of three independent biological replicates. (C) Proteasomes accumulate in autophagic bodies within the vacuole upon nitrogen starvation, but not fixed-carbon starvation. Five-day-old seedlings expressing PAG1-GFP, RPN5a-GFP or GFP-ATG8a were grown on +N +C medium and then switched to –N or –C media and treated with 1 µM ConA for 16 hr before imaging of the root lower elongation zone by confocal fluorescence microscopy. Scale bar, 10 µm. (D) Proteasomes assemble into large cytoplasmic PSG-like structures upon fixed-carbon starvation, instead of the smaller vacuolar puncta seen upon nitrogen starvation. Five-day-old seedlings expressing PAG1-GFP, RPN5a-GFP or GFP-ATG8a were grown, treated and imaged as in panel C, but focusing on cells closer to the root tip. Scale bar, 2 µm. (E) The PSG-like structures that form upon fixed-carbon starvation are not decorated with ATG8a. Roots from 5-day-old seedlings expressing PAG1-GFP and mCherry-ATG8a were grown, treated and imaged as in panel C. Shown are the GFP, mCherry and merged fluorescence channels. Scale bar, 5 µm. (F) The accumulation of PSG-like structures upon fixed-carbon starvation is rapidly reversible upon replenishment of the carbon source. Roots from 5 day-old seedlings expressing PAG1-GFP were grown on +N +C medium, switched to –C medium for 16 hr, and then returned to +N +C medium for the indicated times before imaging as in panel C. Scale bar, 10 µm. In panels C, D, E and F: N, nucleus; V, vacuole; P, PSG.
Figure 9—source data 1. Source data for Figure 9B.