Figure 10. The formation of PSG-like structures in Arabidopsis upon fixed-carbon starvation requires the Blm10 ortholog PA200 and is independent of autophagy.

(A) Elimination of PA200 accelerates proteaphagy of the CP, but not the RP, in response to fixed-carbon starvation. PAG1:PAG1-GFP pag1-1 and RPN5a:RPN5a-GFP rpn5a-2 seedlings with or without the pa200-2 or pa200-3 mutations 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 indicate the GFP fusion and free GFP, respectively. Immunodetection of histone H3 was used to confirm near equal protein loading. (B) Quantification of the free GFP/GFP fusion ratios of the PAG1-GFP and RPN5a-GFP reporters in wild-type (WT), pa200-2 or pa200-3 seedlings upon switching to –C medium. 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) PAG1-GFP fails to coalesce into cytoplasmic PSG-like structures upon fixed-carbon starvation in the absence of PA200, and instead appears in vacuolar autophagic bodies. Five-day-old PAG1:PAG1-GFP pag1-1 seedlings with or without the pa200-2 or pa200-3 mutations were grown on +N +C medium and then transferred to –C medium containing 1 µM ConA and subjected to darkness for 16 hr. Cells were imaged by confocal fluorescence microscopy. Scale bar, 2 µm. (D) The cytoplasmic PSG-like structures containing PAG1-GFP form independently of autophagy. PAG1:PAG1-GFP pag1-1 seedlings with or without the atg7-2 mutation were grown on +N +C medium and then transferred to –N or –C media (in the light or dark, respectively) containing 1 µM ConA for 16 hr. Cells were imaged by confocal fluorescence microscopy as in panel (C). Scale bar, 10 µm. In panels C and D: N, nucleus; V, vacuole; P, PSG.

Figure 10—figure supplement 1. Cytosolic PSG-like structures form in Arabidopsis in the absence of concanamycin A (ConA) treatment.

Proteasomes accumulate in PSG-like foci upon fixed-carbon starvation even in the absence of treatment with ConA, which is required to stabilize autophagic bodies within the vacuole. Five-day-old PAG1:PAG1-GFP pag1-1 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 carbon (–C) for 24 hr before imaging of the root lower elongation zone by confocal fluorescence microscopy. Whereas PSG-like foci are observed in the cytosol upon fixed-carbon starvation, vacuolar autophagic bodies are not observed upon nitrogen starvation due to their rapid degradation in the vacuole in the absence of ConA. Scale bar, 10 µm.