Autophagosome development and chloroplast segmentation occur synchronously for piecemeal degradation of chloroplasts

  1. Masanori Izumi  Is a corresponding author
  2. Sakuya Nakamura
  3. Kohei Otomo
  4. Hiroyuki Ishida
  5. Jun Hidema
  6. Tomomi Nemoto
  7. Shinya Hagihara
  1. Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Japan
  2. Center for Sustainable Resource Science (CSRS), RIKEN, Japan
  3. Exploratory Research Center on Life and Living Systems (ExCELLs), National Institutes of Natural Sciences, Japan
  4. National Institute for Physiological Sciences, National Institutes of Natural Sciences, Japan
  5. The Graduate University for Advanced Studies, SOKENDAI, Japan
  6. Research Institute for Electronic Science, Hokkaido University, Japan
  7. Graduate School of Medicine, Juntendo University, Japan
  8. Graduate School of Agricultural Science, Tohoku University, Japan
  9. Graduate School of Life Sciences, Tohoku University, Japan
10 figures, 20 videos, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Chloroplast buds are released in sugar-starved leaves.

Time-lapse observations of three-dimensional (3D) reconstructed chloroplast morphology in Arabidopsis mesophyll cells accumulating chloroplast stroma–targeted fluorescent markers. A leaf from a …

Figure 1—figure supplement 1
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. Second rosette leaves of 21-day-old plants were excised and incubated …

Figure 1—figure supplement 2
The release of chloroplast buds by autophagy contributes to the decline in chloroplast stroma volume.

(A) Orthogonal projections produced from z-stack images (30 µm in depth) of mesophyll cells from wild-type (WT) or atg7 leaves accumulating the chloroplast stroma marker RBCS-mRFP. Second rosette …

Figure 2 with 2 supplements
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 …

Figure 2—figure supplement 1
Chloroplast buds contain stroma and envelope proteins.

Second rosette leaves accumulating stromal RBCS-GFP along with outer envelope membrane–bound TOC64-mRFP (A), inner envelope membrane–bound KEA1-mRFP (B), or thylakoid membrane–bound ATPC1-tagRFP (C) …

Figure 2—figure supplement 2
A released chloroplast bud contains an inner envelope marker protein.

Time-lapse observations of Arabidopsis mesophyll cells accumulating the chloroplast stroma marker RBCS-GFP along with inner envelope membrane–bound KEA1-mRFP. The second rosette leaf from a …

Figure 3 with 1 supplement
Tracking the transport of a Rubisco-containing body (RCB).

A leaf accumulating chloroplast stroma–targeted GFP (CT-GFP) was incubated in sugar-free solution in darkness for 6 hr from dawn, and the transport of an RCB was tracked. The second rosette leaf …

Figure 3—figure supplement 1
Rubisco-containing bodies (RCBs) appear to begin random movement during their tracking.

A leaf accumulating RBCS-tagRFP (A) or RBCS-EYFP (B) was incubated in sugar-free solution in the dark for 7–8 hr from dawn, and the transport of an RCB was tracked. Second rosette leaves from …

Figure 4 with 1 supplement
Dynamics of the vacuolar membrane during the incorporation of Rubisco-containing bodies (RCBs).

Leaves accumulating the chloroplast stroma marker RBCS-mRFP along with the vacuolar membrane marker VHP1-mGFP were incubated in sugar-free solution in darkness for 6–8 hr from dawn, and the behavior …

Figure 4—figure supplement 1
Additional observations of the incorporation of Rubisco-containing bodies (RCBs) into the vacuolar lumen.

Additional time-lapse data of the experiments described in Figure 4. The second rosette leaves from 21- (A) or 24-day-old (B, C) plants were used. The images in (A), (B) and (C) are still frames …

Figure 5 with 1 supplement
Autophagy deficiency does not increase the number of chloroplast protrusions during a 1-day 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 day in …

Figure 5—figure supplement 1
Chloroplast protrusions do not increase in atg2 or atg10 mutant leaves during a 1-day 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. Second rosette leaves from 21-day-old …

Figure 6 with 4 supplements
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 for 6 hr from dawn (A) …

Figure 6—figure supplement 1
Another observation of the budding of the isolation membrane–associated site in a chloroplast.

(A) A leaf accumulating the chloroplast stroma marker CT-DsRed and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in darkness for 6 hr from dawn and then observed. A …

Figure 6—figure supplement 2
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 was incubated in sugar-free solution in darkness for 5 hr from dawn and then observed. A …

Figure 6—figure supplement 3
Proportion of chloroplast buds engulfed by GFP-ATG8a-labeled isolation membranes.

Leaves accumulating the chloroplast stroma marker RBCS-mRFP and the isolation membrane marker GFP-ATG8a were incubated in sugar-free solution in the dark for 5–9 hr from dawn, and chloroplast …

Figure 6—figure supplement 4
Proportion of GFP-ATG8a-labeled structures that are associated with chloroplasts.

Leaves accumulating the chloroplast stroma marker RBCS-mRFP and the isolation membrane marker GFP-ATG8a were incubated in sugar-free solution in the dark for 5–9 hr from dawn, and GFP-ATG8a-labeled …

Figure 7 with 3 supplements
Diminished salicylic acid signal suppresses stromule formation in autophagy-deficient mutants.

(A) Orthogonal projections produced from z-stack images (10 μm in depth) of guard cells from wild-type (WT), atg5, NahG, and atg5 NahG leaves accumulating chloroplast stroma−targeted GFP (CT-GFP). …

Figure 7—figure supplement 1
Autophagy deficiency activates stromule formation from mesophyll chloroplasts accumulating RBCS-mRFP in senescing leaves.

(A) Orthogonal projections produced from z-stack images (15 µm in depth) of mesophyll cells from leaves of wild-type (WT), atg5, and atg7 plants accumulating RBCS-mRFP. Third rosette leaves from …

Figure 7—figure supplement 2
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 wild-type (WT), atg5, atg7, sid2, sid2 atg5, and sid2 atg7 leaves accumulating CT-GFP. Third rosette …

Figure 7—figure supplement 3
NahG expression does not counteract the increase in hydrogen peroxide (H2O2) content produced by the atg5 mutation.

H2O2 contents in third rosette leaves from 36-day-old plants. Wild-type (WT) plants without any transgenic construct and CT-GFP-expressing plants in the WT, atg5, NahG, and atg5 NahG backgrounds …

Figure 8 with 2 supplements
DRP5B is dispensable for chloroplast autophagy in sugar-starved leaves.

(A) Confocal images of mesophyll cells from wild-type (WT) and drp5b leaves accumulating the stroma marker RBCS-mRFP. Second rosette leaves from 21-day-old plants were incubated in sugar-free …

Figure 8—source data 1

Original files for western blot analysis displayed in Figure 8C.

https://cdn.elifesciences.org/articles/93232/elife-93232-fig8-data1-v1.zip
Figure 8—source data 2

PDF file containing original western blots for Figure 8C, indicating the relevant bands, genotypes, and conditions.

https://cdn.elifesciences.org/articles/93232/elife-93232-fig8-data2-v1.zip
Figure 8—source data 3

Source data for the graphs in Figure 8.

https://cdn.elifesciences.org/articles/93232/elife-93232-fig8-data3-v1.zip
Figure 8—figure supplement 1
Production of Rubisco-containing bodies (RCBs) 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 from 21-day-old plants were incubated in …

Figure 8—figure supplement 2
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. Second rosette leaves from 21-day-old plants were incubated in …

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 for 5 hr from dawn and then observed. Arrowheads indicate a chloroplast …

Author response image 1
Mesophyll cells in a leaf of atg7 accumulating stromal CT-GFP, reconstructed from the data shown in the previous version of Figure 7–figure supplement 1.

(A) Individual channel images (CT-GFP and chlorophyll) from the merged orthogonal projection image shown in the previous version of Figure 7–figure supplement 1. The right panel shows the enhanced …

Videos

Video 1
Release of a chloroplast bud as visualized by chloroplast stroma–targeted GFP.

The second rosette leaf from a 21-day-old plant accumulating the chloroplast stroma–targeted GFP (CT-GFP) was incubated in sugar-free solution in darkness for time-lapse imaging with a two-photon …

Video 2
Release of a chloroplast bud as visualized by RBCS-mRFP.

The second rosette leaf from a 22-day-old plant accumulating the chloroplast stroma marker RBCS-mRFP was incubated in sugar-free solution in darkness for time-lapse imaging with a two-photon …

Video 3
Tracking of the stroma marker and chlorophyll fluorescence during the release of a chloroplast bud.

The second rosette leaf from a 20-day-old plant accumulating the chloroplast stroma marker RBCS-EYFP was incubated in sugar-free solution in darkness for time-lapse imaging with a two-photon …

Video 4
A released chloroplast bud contains the outer envelope marker TOC64-mRFP.

The second rosette leaf from a 21-day-old plant accumulating the chloroplast stroma marker RBCS-GFP and the outer envelope marker TOC64-mRFP was incubated in sugar-free solution in darkness for …

Video 5
A released chloroplast bud contains the inner envelope marker KEA1-mRFP.

The second rosette leaf from a 22-day-old plant accumulating the chloroplast stroma marker RBCS-GFP and the inner envelope marker KEA1-mRFP was incubated in sugar-free solution in the dark for …

Video 6
A released chloroplast bud does not contain the thylakoid membrane marker ATPC1-tagRFP.

The second rosette leaf from a 21-day-old plant accumulating the chloroplast stroma marker RBCS-GFP and the thylakoid membrane marker ATPC1-tagRFP was incubated in sugar-free solution in darkness …

Video 7
Tracking of a Rubisco-containing body (RCB) marked by CT-GFP.

The second rosette leaf from a 21-day-old plant accumulating chloroplast stroma–targeted GFP (CT-GFP) was incubated in sugar-free solution in darkness for time-lapse monitoring of an RCB marked by …

Video 8
Tracking of a Rubisco-containing body (RCB) marked by RBCS-tagRFP.

The second rosette leaf from a 22-day-old plant accumulating stromal RBCS-tagRFP was incubated in sugar-free solution in the dark for time-lapse monitoring of an RCB marked by RBCS-tagRFP. Arrowhead …

Video 9
Tracking of a Rubisco-containing body (RCB) marked by RBCS-EYFP.

The second rosette leaf from a 22-day-old plant accumulating stromal RBCS-EYFP was incubated in sugar-free solution in the dark for time-lapse monitoring of an RCB marked by RBCS-EYFP. Arrowhead …

Video 10
Incorporation of a Rubisco-containing body (RCB) into the vacuolar lumen.

The second rosette leaf from a 21-day-old plant accumulating the chloroplast stroma marker RBCS-mRFP along with the vacuolar membrane marker VHP1-mGFP was incubated in sugar-free solution in …

Video 11
Video 2 of the vacuolar incorporation of a Rubisco-containing body (RCB).

Additional time-lapse data for the experiment described in Figure 4 and Video 10. The second rosette leaf from a 23-day-old plant was used. Arrowhead indicates an RCB incorporated into the vacuolar …

Video 12
Video 3 of the vacuolar incorporation of a Rubisco-containing body (RCB).

Additional time-lapse data for the experiment described in Figure 4 and Video 10. Arrowhead indicates an RCB incorporated into the vacuolar lumen. Images acquired every 1.5 s are displayed at 10 …

Video 13
Video 4 of the vacuolar incorporation of a Rubisco-containing body (RCB).

Additional time-lapse data for the experiment described in Figure 4 and Video 10. Arrowhead indicates an RCB incorporated into the vacuolar lumen. Images acquired every 1.5 s are displayed at 10 …

Video 14
Video 5 of the vacuolar incorporation of a Rubisco-containing body (RCB).

Additional time-lapse data for the experiment described in Figure 4 and Video 10. Arrowhead indicates an RCB incorporated into the vacuolar lumen. Images acquired every 1.5 s are displayed at 10 …

Video 15
Budding of the isolation membrane–associated site within a chloroplast.

The second rosette leaf from a 23-day-old plant accumulating the chloroplast stroma marker RBCS-mRFP and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in darkness for …

Video 16
Autophagosome development and chloroplast segmentation occur concomitantly.

The second rosette leaf from a 23-day-old plant accumulating the chloroplast stroma–targeted DsRed (CT-DsRed) and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in …

Video 17
Another time-lapse assay showing the concomitant progression of autophagosome development and chloroplast segmentation.

The second rosette leaf from a 22-day-old plant accumulating the chloroplast stroma–targeted DsRed (CT-DsRed) and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in …

Video 18
Autophagy-related chloroplast segmentation occurs in sequence.

The second rosette leaf from a 21-day-old plant accumulating the chloroplast stroma marker RBCS-tagRFP and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in darkness …

Video 19
Release of a chloroplast bud in a sugar-starved leaf of the drp5b mutant.

The second rosette leaf from a 21-day-old drp5b plant accumulating the chloroplast stroma marker RBCS-mRFP was incubated in sugar-free solution in darkness for time-lapse imaging. Arrowhead …

Video 20
An enlarged chloroplast caused by the drp5b mutation forms Rubisco-containing bodies (RCBs) along the isolation membrane−associated sites.

The second rosette leaf from a 22-day-old drp5b plant accumulating the chloroplast stroma marker RBCS-mRFP and the isolation membrane marker GFP-ATG8a was incubated in sugar-free solution in …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Arabidopsis thaliana)RBCS2BTAIRAT5G38420
Gene (A. thaliana)VHP1TAIRAT1G15690
Gene (A. thaliana)TOC64-IIITAIRAT3G17970
Gene (A. thaliana)ATG8aTAIRAT4G21980
Gene (A. thaliana)ATPC1TAIRAT4G04640
Gene (A. thaliana)KEA1TAIRAT1G01790
Genetic reagent (A. thaliana)atg5-1ABRCSAIL_129_B07
Genetic reagent (A. thaliana)atg7-2ABRCGABI_655B06
Genetic reagent (A. thaliana)atg2-1ABRCSALK_076727
Genetic reagent (A. thaliana)atg10-1ABRCSALK_084434
Genetic reagent (A. thaliana)drp5b (arc5-2)ABRCSAIL_71_D11
Genetic reagent (A. thaliana)sid2-2ABRCCS16438
Genetic reagent (A. thaliana)NahG atg5-110.1105/tpc.109.068635
Genetic reagent (A. thaliana)sid2-2 atg5-110.1105/tpc.109.068635
Genetic reagent (A. thaliana)Pro35S:CT-GFP10.1126/science.276.5321.2039
Genetic reagent (A. thaliana)Pro35S:CT-DsRed10.1093/pcp/pcab084
Genetic reagent (A. thaliana)ProRBCS:RBCS-GFP10.1104/pp.108.122770
Genetic reagent (A. thaliana)ProVHP1:VHP1-mGFP10.1105/tpc.114.127571
Genetic reagent (A. thaliana)ProUBQ:GFP-ATG8a10.1093/pcp/pcaa162
Genetic reagent (A. thaliana)ProTOC64:TOC64-mRFP10.26434/chemrxiv-2023-kx6gp
Genetic reagent (A. thaliana)ProRBCS:RBCS-EYFPThis paperSee ‘Plant materials’ in Materials and methods
Genetic reagent (A. thaliana)ProRBCS:RBCS-mRFPThis paperSee ‘Plant materials’ in Materials and methods
Genetic reagent (A. thaliana)ProRBCS:RBCS-tagRFPThis paperSee ‘Plant materials’ in Materials and methods
Genetic reagent (A. thaliana)ProATPC1:ATPC1-tagRFPThis paperSee ‘Plant materials’ in Materials and methods
Genetic reagent (A. thaliana)ProKEA1:KEA1-mRFPThis paperSee ‘Plant materials’ in Materials and methods
AntibodyAnti-RFP (Mouse, monoclonal)MBLM204-3(1:2000)
AntibodyAnti-cFBPase (Rabbit, polyclonal)AgriseraAS04043(1:5000)
Recombinant DNA reagentProRBCS:RBCS-EYFPThis paperSee ‘Plant materials’ in Materials and methods
Recombinant DNA reagentProRBCS:RBCS-mRFPThis paperSee ‘Plant materials’ in Materials and methods
Recombinant DNA reagentProRBCS:RBCS-tagRFPThis paperSee ‘Plant materials’ in Materials and methods
Recombinant DNA reagentProATPC1:ATPC1-tagRFPThis paperSee ‘Plant materials’ in Materials and methods
Recombinant DNA reagentProKEA1:KEA1-mRFPThis paperSee ‘Plant materials’ in Materials and methods
Sequence-based reagentATPC1_FThis paperPCR primers (cloning)CACCCATGGAGAGGGCTCGTACCTTAC
Sequence-based reagentATPC1_RThis paperPCR primers (cloning)AACCTGTGCATTAGCTCCAG
Sequence-based reagentKEA1_FThis paperPCR primers (cloning)AGGAACCAATTCAGTCGACTCATGATCATAACAAGTCTC
Sequence-based reagentKEA1_RThis paperPCR primers (cloning)AAAGCTGGGTCTAGATATCCGATTACGACTGTGCCTCCTTC
Commercial assay or kitAmplex Red Hydrogen Peroxide/Peroxidase Assay KitInvitrogenA22188
Chemical compound, drugConcanamycin ASanta Cruzsc-202111
Software, algorithmZENCarl ZeissRRID:SCR_013672Image processing and quantification (microscopy)
Software, algorithmNIS-Elements CNikonRRID:SCR_020318Image processing and quantification (microscopy)
Software, algorithmLAS XLeicaRRID:SCR_013673Image processing and quantification (microscopy)
Software, algorithmImarisBitplaneRRID:SCR_007370Image processing and quantification (microscopy)
Software, algorithmImage labBio-RadRRID:SCR_014210Image processing and quantification (western blot)
Software, algorithmJMP14SASRRID:SCR_022199Statistics

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