Chloroplast buds are released in sugar-starved leaves.

Time-lapse observations of 3D-reconstructed chloroplast morphology in Arabidopsis mesophyll cells accumulating chloroplast stroma-targeted fluorescent markers. A leaf from a plant accumulating chloroplast stroma-targeted GFP (CT-GFP) (A), RBCS-mRFP (B), or RBCS-EYFP (C) was incubated in sugar-free solution in darkness and then observed through a two-photon excitation microscope equipped with a confocal spinning disk unit. Images in (A–C) are still frames from videos 1, 2, and 3, respectively. Arrowheads indicate chloroplast budding structures. Scale bars, 5 µm. In (C), green, RBCS-EYFP; magenta, chlorophyll fluorescence. In the merged images, the overlapping regions of RBCS-EYFP and chlorophyll signals appear white.

Chloroplast buds containing stroma and envelope components are released from the chloroplasts.

Time-lapse observations of Arabidopsis mesophyll cells accumulating the chloroplast stroma marker along with an envelope marker or a thylakoid membrane marker. Leaves accumulating stromal RBCS-GFP along with envelope-bound TOC64-mRFP (A and B) or with thylakoid membrane-bound ATPC1-tagRFP (C and D) were incubated in sugar-free solution in darkness and then observed. Images in (B) or (D) are still frames from Videos 4 and 5, respectively. Arrowheads indicate chloroplast budding structures. Scale bars, 5 µm. Green, TOC64-mRFP or ATPC1-tagRFP; magenta, RBCS-GFP; orange, chlorophyll (Chl) fluorescence. The graphs in (A and C) show fluorescence intensities along the blue lines (a to b) in the magnified images of the area indicated by dashed blue boxes. The intensities are shown relative to the maximum intensity for each fluorescence channel, set to 1.

Tracking the transport of a Rubisco-containing body.

A leaf accumulating chloroplast-stroma targeted GFP (CT-GFP) was incubated in sugar-free solution in darkness and the transport of a Rubisco-containing body (RCB) was tracked. (A) Confocal images during the periods when the RCB moved quickly (24.4–34.8 and 73.8–90.5 sec). Arrowheads indicate an RCB. The images were generated from Video 6. Green, CT-GFP; magenta, chlorophyll (Chl) fluorescence. In the merged images, the overlapping regions of RBCS-GFP and chlorophyll signals appear white. Scale bar, 5 µm. (B) Calculated speed of the tracked RCB in (A). (C) The track of the RCB. The color of the track line changes over time, as indicated by the color bar. Scale bar, 2 µm.

Dynamics of the vacuolar membrane during the incorporation of Rubisco-containing bodies.

Leaves accumulating the chloroplast stroma marker RBCS-mRFP along with the vacuolar membrane marker VHP1-mGFP were incubated in sugar-free solution in darkness and the behavior of cytosolic RCBs was monitored. The images when the vacuolar membrane engulfs an RCB (25.4–30.5 sec in A and 52.6–57.4 sec in B) and when an RCB is incorporated into the vacuolar lumen (47.0–52.0 sec in A and 65.3–72.9 in B) are shown. The images in (A) and (B) are still frames from Videos 7 and 8, respectively. Open arrowheads indicate an RCB engulfed by the vacuolar membrane. Closed arrowheads indicate the open site of the vacuolar membrane for the incorporation of an RCB. V indicates the region of the vacuolar lumen. Green, VHP1-mGFP; magenta, RBCS-mRFP. Scale bars, 5 µm.

Autophagy deficiency does not increase the number of chloroplast protrusions during a 1-d dark treatment.

Leaves from wild-type (WT), atg5, or atg7 plants accumulating the chloroplast stroma marker RBCS-mRFP were incubated in sugar-free solution containing 1 µM concanamycin A (concA) for 1 d in darkness. 2D images of mesophyll cells were acquired (A), and the number of accumulated RCBs in the vacuoles was scored (B). Leaves from untreated plants are shown as control. The appearance of chloroplast protrusions was observed from orthogonal projections created from z-stack images (15 µm in depth; C), and the proportion of chloroplasts having protrusion structures was calculated (D). Scale bars, 10 µm. Green, RBCS-mRFP; magenta, chlorophyll fluorescence. Only the merged channels are shown. The overlapping regions of RBCS-mRFP and chlorophyll signals appear white. Small vesicles containing RBCS-mRFP without chlorophyll signal appear as green and are RCBs in the vacuole. Arrowheads indicate the structures that were counted as a chloroplast protrusion in (D). Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 4). Dots represent individual data points in each graph.

The formation of a chloroplast bud and the maturation of the chloroplast-associated isolation membrane occur concomitantly.

Leaves accumulating the chloroplast stroma marker, RBCS-mRFP (A) or CT-DsRed (B), and the isolation membrane marker GFP-ATG8a were incubated in sugar-free solution in darkness and then observed. Arrowheads indicate the position of the chloroplast-associated isolation membrane. Images in (A) and (B) are still frames from Videos 9 and 10, respectively. Green, GFP-ATG8a; magenta, RBCS-mRFP or CT-DsRed. Scale bars, 5 µm. (C) Time-dependent changes in the ratio of the major axis to the minor axis in the GFP-ATG8a-labeled isolation membrane (top), or in the area of the chloroplast protrusion (bottom) as measured from the images in (B).

Diminished salicylic acid signal suppresses stromule formation in autophagy-deficient mutants.

(A) Confocal images of guard cells from wild-type (WT), atg5, NahG, and atg5 NahG leaves accumulating chloroplast stroma-targeted GFP (CT-GFP). Scale bars, 10 µm. (B) Percentage of chloroplasts forming stromules, from the observations described in (A). (C) Confocal images of guard cells from WT, atg5, atg7, sid2, sid2 atg5, and sid2 atg7 leaves accumulating CT-GFP. Scale bars, 10 µm. (D) Percentage of chloroplasts forming stromules, from the observations described in (C). In (A) and (C), orthogonal projections produced from z-stack images (10 μm in depth) are shown. (E) Orthogonal projections produced from z-stack images (15 µm in depth) of mesophyll cells from WT, atg5, atg7, sid2, sid2 atg5, and sid2 atg7 leaves accumulating CT-GFP. Third rosette leaves from 36-d-old plants were observed. Green, CT-GFP; magenta, chlorophyll fluorescence. Only the merged channels are shown. Scale bars, 20 µm. (F) Percentage of chloroplasts forming stromules in mesophyll cells, from the observations described in (E). (G) Hydrogen peroxide (H2O2) content in WT, atg5, atg7, sid2, sid2 atg5, and sid2 atg7 leaves. Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 3 in B and D or 4 in F and G). Dots represent individual data points in each graph.

DRP5b is dispensable for chloroplast autophagy in sugar-starved leaves.

Confocal images of mesophyll cells from wild-type (WT) and drp5b leaves accumulating the stroma marker RBCS-mRFP. Second rosette leaves were incubated in sugar-free solution containing 1 µM concanamycin A (concA) in darkness. Green, RBCS-mRFP; magenta, chlorophyll fluorescence. Scale bars, 10 µm. (B) Number of accumulated RCBs in WT, drp5b, atg5, and atg7 leaves, counted from the observations described in (A). Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 4–5). (C) Biochemical detection of autophagy flux for chloroplast stroma based on a free RFP assay. RFP and cFBPase (loading control) were detected by immunoblotting of soluble protein extracts from leaves of WT, drp5b, atg5, and atg7 plants accumulating RBCS-mRFP. Protein extracts from either untreated control leaves (cont) or leaves after 2 d of incubation in darkness (dark) were used. Total protein was detected by Coomassie Brilliant Blue (CBB) staining as a loading control. The filled arrowhead indicates RBCS-mRFP fusion, and the open arrowhead indicates free mRFP derived from the cleavage of RBCS-mRFP. (D) Quantification of the free mRFP/RBCS-mRFP ratio shown relative to that of untreated wild-type plants, which was set to 1. Asterisks denote significant differences based on t-test (***, P < 0.001; n.s., not significant). Values are means ±SE (n = 4). Dots represent individual data points in each graph.

Formation and segmentation of chloroplast buds in leaves of the drp5b mutant.

(A) A leaf from the drp5b mutant accumulating the stroma marker RBCS-mRFP was incubated in sugar-free solution in darkness and then observed. Arrowheads indicate a chloroplast bud. Green, RBCS-mRFP; magenta, chlorophyll fluorescence (Chl). (B) A leaf from the drp5b mutant accumulating the stroma marker RBCS-mRFP and the isolation membrane marker GFP-ATG8a were incubated in sugar-free solution in darkness and then observed. Arrowheads indicate the position of the chloroplast-associated isolation membrane. White or blue arrowheads indicate different isolation-membrane-associated sites in a chloroplast, respectively. Green, GFP-ATG8a; magenta, RBCS-mRFP; orange, chlorophyll fluorescence (Chl). Scale bars, 5 µm.

Accumulation of chloroplast stroma components in the vacuole via autophagy.

Confocal images of mesophyll cells from wild-type (A) or atg7 (B) plants accumulating the chloroplast stroma marker RBCS-mRFP. The excised leaves were incubated in 10 mM MES-NaOH containing 1 µM concanamycin A (concA) in darkness. Sucrose (Suc) or Murashige and Skoog salts (MS) were added as an energy or nutrient source, respectively. Second rosette leaves from nontreated plants are shown as control. Green, RBCS-mRFP; magenta, chlorophyll fluorescence. Scale bars, 10 µm. The small vesicles containing RBCS-mRFP without chlorophyll signal appear as green and are Rubisco-containing bodies (RCBs) in the vacuole. (C) Number of accumulated RCBs from the observations described in (A) and (B). Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 4). Dots represent individual data points.

Chloroplast protrusions do not increase in atg2 or atg10 mutant leaves during a 1-d dark treatment.

The experiments described in Figure 5 were performed on leaves from wild-type (WT), atg2, or atg10 plants accumulating the chloroplast stroma marker RBCS-mRFP. Scale bars, 10 µm. Green, RBCS-mRFP; magenta, chlorophyll fluorescence. Only the merged channels are shown. The overlapping regions of RBCS-mRFP and chlorophyll signals appear white. Small vesicles containing RBCS-mRFP without chlorophyll signal appear as green and are RCBs in the vacuole. Arrowheads indicate the structures that were counted as a chloroplast protrusion in (D). Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 4). Dots represent individual data points in each graph.

Another observation of the protrusion of the isolation-membrane-associated site in a chloroplast.

A leaf accumulating the chloroplast stroma marker CT-DsRed and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in darkness and then observed. Arrowheads indicate the position of the chloroplast-associated isolation membrane. The images are still frames from Video 11. Green, GFP-ATG8a; magenta, CT-DsRed. Scale bars, 5 µm. (B) Time-dependent changes in the ratio of the major axis to the minor axis in the GFP-ATG8a-labeled isolation membrane (top), or in the area of the chloroplast protrusion (bottom), as measured from the images in (A).

Chloroplast buds surrounded by the isolation membrane appear in multiple chloroplasts.

A leaf accumulating the chloroplast stroma marker RBCS-tagRFP and the isolation membrane marker GFP-ATG8a were incubated in sugar-free solution in darkness and then observed. White, yellow, or blue arrowheads indicate the isolation-membrane-associated site in different chloroplasts, respectively. The images are still frames from Video 12. Green, GFP-ATG8a; magenta, RBCS-tagRFP; orange, chlorophyll (Chl) fluorescence. Scale bars, 5 µm.

Autophagy deficiency does not activate stromule formation from mesophyll chloroplasts in young leaves.

Orthogonal projections produced from z-stack images (15 µm in depth) of mesophyll cells from WT, atg5, atg7, sid2, sid2 atg5, and sid2 atg7 leaves accumulating CT-GFP. The third rosette leaves from 20-d-old plants were observed. Green, CT-GFP; magenta, chlorophyll fluorescence. Only the merged channels are shown. Scale bars, 20 µm.

Production of Rubisco-containing bodies in drp5b mutants is ATG5-dependent.

(A) Confocal images of mesophyll cells from wild-type (WT), atg5, drp5b, and drp5b atg5 leaves accumulating the stroma marker CT-GFP. Second rosette leaves were incubated in sugar-free solution containing 1 µM concanamycin A (concA) in darkness. Second rosette leaves from untreated plants were used as control. Green, CT-GFP; magenta, chlorophyll fluorescence. Only the merged channels are shown. Small vesicles containing CT-GFP without chlorophyll signal appear as green and are RCBs in the vacuole. Scale bars, 10 µm. (B) Number of accumulated RCBs, counted from the observations described in (A). Different lowercase letters denote significant differences based on Tukey’s test (P < 0.05). Values are means ±SE (n = 5). Dots represent individual data points.

Vacuolar accumulation of stromal marker proteins in sugar-starved leaves.

(A) Confocal images of mesophyll cells from wild-type (WT), drp5b, atg5, and atg7 leaves accumulating the stroma marker RBCS-mRFP. The leaves were incubated in sugar-free solution in darkness for 2 d. Second rosette leaves from untreated plants are shown as control. Green, RBCS-mRFP; magenta, chlorophyll fluorescence. Scale bars, 20 µm. (B) RFP intensity in the vacuolar lumen, as measured from the observations described in (A) and shown relative to that of untreated WT plants, which was set to 1. Asterisks denote significant differences based on t-test (***, P < 0.001; n.s., not significant). Values are means ±SE (n = 4). Dots represent individual data points.