The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts
- Center for Quantitative Cell Imaging, University of Wisconsin-Madison, United States
- Department of Botany, University of Wisconsin-Madison, United States
- Department of Biology, Washington University in St. Louis, United States
- ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Australia
- Department of Plant Biology,School of Agronomy, Universidad de la República, Uruguay
- Department of Medical Physics, University of Wisconsin-Madison, United States
- Department of Biomedical Engineering, University of Wisconsin-Madison, United States
- Morgridge Institute for Research, United States
Figures
Figure 1
NBR1 associates with chloroplasts after HL exposure.
(A) Confocal imaging of NBR1-GFP and chlorophyll autofluorescence in cotyledons and hypocotyl cells of 8-day-old wild-type seedlings grown under low light (LL, 40 μmol m–2 s–1) or left to recover …
Figure 2 with 1 supplement
Ultrastructure of chloroplasts in wild-type, atg7, and nbr1 cotyledons 24 hr after HL exposure.
(A) Transmission electron micrograph of a high-pressure frozen/freeze-substituted atg7 cotyledon mesophyll cell from 8-day-old seedlings exposed to HL and left to recover for 24 hr. Three different …
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Figure 2—source data 1
G.Original files of full raw unedited blots and figure with uncropped blots.
- https://cdn.elifesciences.org/articles/86030/elife-86030-fig2-data1-v2.zip
Figure 2—figure supplement 1
Uncropped immunoblot of total proteins from wild type plants expressing NBR1-GFP, and atg7-1 and nbr1 mutants using anti-NBR1 antibodies (Jung et al., 2020).
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Figure 2—figure supplement 1—source data 1
Original files of full raw unedited blots.
Uncropped blots are shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/86030/elife-86030-fig2-figsupp1-data1-v2.zip
Figure 3
Recruitment of NBR1 and ATG8A to photodamaged chloroplasts.
(A) Confocal imaging of cotyledon mesophyll cells from 8-day-old seedlings expressing mCherry-NBR1 and GFP-ATG8A under LL (top) and at 24 hr after HL exposure (bottom). Magenta, cyan, and white …
Figure 4
Vacuolar delivery of NBR1-positive chloroplast into the vacuole.
(A) Projection of three confocal images (z1–z3) and several other confocal images of cotyledon mesophyll cells from 1-week-old, wild-type seedlings expressing the tonoplast marker YFP-VAMP711 and …
Figure 5
Chloroplast remodeling after HL exposure.
(A) Projections of 20 confocal images along a z-stack taken from the adaxial side of cotyledon mesophyll cells from 8-day-old wild-type (WT), atg7, and nbr1 seedlings expressing RECA-GFP. Seedlings …
Figure 6 with 2 supplements
Chloroplast proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
(A) Volcano plots showing the changes in the relative abundance of chloroplast proteins under LL or HL, in wild type (WT) and mutants. The number on the top of each plot indicates the total number …
Figure 6—figure supplement 1
Proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
(A) Volcano plots showing the relative abundance changes of total proteins detected in wild type (Col-0), nbr1, atg7, and nbr1 atg7 seedlings grown either under LL or exposed to HL and let recover …
Figure 6—figure supplement 2
Proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of chloroplast proteins localized to envelopes, stroma, and thylakoid membranes.
Volcano plots showing the relative abundance changes of chloroplast proteins grouped by subcompartments (envelopes, stroma, and thylakoids) in wild-type (Col-0), nbr1, atg7, and nbr1 atg7 seedlings …
Figure 7 with 1 supplement
NBR1 domains have distinct roles in recruiting NBR1 to chloroplasts after HL treatment.
(A) Diagram of the Arabidopsis NBR1 protein and its domains. FW, Four-Tryptophan domain; PB1, Phox and Bem1p domain; ZZ, ZZ-type zinc finger domain; UBA1 and UBA2, ubiquitin-associated domains; AIM, …
Figure 7—figure supplement 1
NBR1 domains in NBR1 recruitment to chloroplasts in nbr1 atg7 double mutant cotyledons after HL treatment.
(A) Confocal imaging of NBR1 mutated proteins fused to YFP expressed in 8-day-old nbr1 atg7 seedlings grown under LL (top) or 24 hr after HL exposure (bottom). Hollow arrowheads and filled …
Figure 8
NBR1 association with chloroplasts in mutants lacking SP1 and PUB4 E3 ligases.
(A) Confocal imaging of NBR1-GFP in 8-day-old wild type (Col-0), sp1, pub4, and sp1 pub4 seedlings under LL and 24 hr after HL exposure. Arrowheads indicate chloroplasts decorated with NBR1-GFP. (B) …
Figure 9
The TIC-TOC translocon is not required for the internalization of NBR1 into photodamaged chloroplasts.
(A) Confocal imaging of NBR1-GFP in wild-type Col-0 (WT) and toc132 cotyledon mesophyll cells from 1-week-old seedlings grown under LL or at 24 hr after HL exposure. Arrowheads indicate chloroplasts …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | NBR1 | AT4G24690 | ||
| Gene (Arabidopsis thaliana) | ATG7 | AT5G45900 | ||
| Gene (Arabidopsis thaliana) | SP1 | AT1G63900 | ||
| Gene (Arabidopsis thaliana) | PUB4 | AT2G23140 | ||
| Gene (Arabidopsis thaliana) | TOC132 | AT2G16640 | ||
| Gene (Arabidopsis thaliana) | TIC40 | AT5G16620 | ||
| Strain, strain background (Arabidopsis thaliana) | Col-0 | |||
| Strain, strain background (Agrobacterium tumefaciens) | GV3101 | |||
| Genetic reagent (Arabidopsis thaliana) | atg7-2 | PMID:20136727 | AT5G45900 | GABI_655B06 |
| Genetic reagent (Arabidopsis thaliana) | nbr1-1 | PMID:23341779 | AT4G24690 | SALK_135513 |
| Genetic reagent (Arabidopsis thaliana) | nbr1-2 | PMID:23341779 | AT4G24690 | GABI_246 H08 |
| Genetic reagent (Arabidopsis thaliana) | toc132-2 | PMID:15273297 | AT2G16640 | SAIL_667_04 |
| Genetic reagent (Arabidopsis thaliana) | tic40-4 | PMID:15659100 | AT5G16620 | SAIL_192_C10 |
| Genetic reagent (Arabidopsis thaliana) | sp1-2 | PMID:23118188 | AT1G63900 | SALK_063571 |
| Genetic reagent (Arabidopsis thaliana) | pub4-2 | PMID:26494759 | AT2G23140 | SALK_054373 |
| Genetic reagent (Arabidopsis thaliana) | Pro35S:mCherry-NBR1 | PMID:21606687 | AT4G24690 | |
| Genetic reagent (Arabidopsis thaliana) | ProUBQ10:mCherry-NBR1 | PMID:21606687 | AT4G24690 | |
| Genetic reagent (Arabidopsis thaliana) | ProNBR1:NBR1-GFP | PMID:28223514, 32967551 | AT4G24690 | |
| Genetic reagent (Arabidopsis thaliana) | Pro35S:RECA-GFP | PMID:9197266, 25649438 | ||
| Genetic reagent (Arabidopsis thaliana) | ProUBQ10:YFP-NBR1 | This study | AT4G24690 | See Methods and Materials Section 1 |
| Genetic reagent (Arabidopsis thaliana) | ProUBQ10:YFP-NBR1mPB | This study | AT4G24690 | See Methods and Materials Section 1 |
| Genetic reagent (Arabidopsis thaliana) | ProUBQ10:YFP-mAIM | This study | AT4G24690 | See Methods and Materials Section 1 |
| Genetic reagent (Arabidopsis thaliana) | ProUBQ10:YFP-NBR1DUBA2 | This study | AT4G24690 | See Methods and Materials Section 1 |
| Antibody | anti-NBR1 (Rabbit polyclonal) | PMID:31494674 | EM IL (1:10) WB (1:1000) | |
| Antibody | anti-TIC40 (Rabbit polyclonal) | Agrisera | Cat#: AS10709 | WB (1:2000) |
| Antibody | anti-PsbA/D1 (Rabbit polyclonal) | Agrisera | Cat#: AS05084 | WB (1:10,000) |
| Antibody | anti-LHCIIb (Rabbit polyclonal) | Agrisera | Cat#: AS01004 | WB (1:5000) |
| Recombinant DNA reagent | ProUBQ10:YFP-NBR | This study | AT4G24690 | See Materials and methods Section 1 |
| Recombinant DNA reagent | ProUBQ10:YFP-NBR1mPB | This study | AT4G24690 | See Materials and methods Section 1 |
| Software, algorithm | CLC main work bench 7 | Qiagen | Cloning | |
| Software, algorithm | Zen Software | Carl Zeiss | Microscopy | |
| Software, algorithm | Image J (Fiji) | NIH | Image Quantification |
Additional files
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Supplementary file 1
Proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
(a) Proteins identified by at least 2 peptide spectral matches.(b) Normalized protein abundances based on the average of two technical replicates or used directly if the proteins were only detected in one technical replicate.(c) Protein abundances expressed as Log2 values.(d) Relative changes of protein abundance between LL and HL conditions in WT plants. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(e) Relative changes of protein abundance between LL and HL conditions in the atg7 mutant. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(f) Relative changes of protein abundance between LL and HL conditions in the nbr1 mutant. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(g) Relative changes of protein abundance between LL and HL conditions in the nbr1 atg7 double mutant. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(h) Comparison of protein abundances between WT and the atg7 mutant under HL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(i) Comparison of protein abundances between WT and the nbr1 mutant under HL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(j) Comparison of protein abundances between WT and the nbr1 atg7 double mutant under HL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(k) Comparison of protein abundances between WT and the atg7 mutant under LL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(l) Comparison of protein abundances between WT and the nbr1 mutant under LL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(m) Comparison of protein abundances between WT and the nbr1 atg7 double mutant under LL conditions. Analysis was performed using the Perseus platform 2.0.6.0 (Tyanova et al., 2016), intensity values from MS/MS were log2 imputed and missing values were replaced with random numbers from a Gaussian distribution with a width of 0.3 and a downshift of 1.8. Statistical significance was determined using t-tests. The protein localizations and functions were categorized based on the GO term listed below. GO:0006914 (Autophagy), GO:0000502 (Proteasome), GO:0009507 (Chloroplast), GO:0005739 (Mitochondria), GO:0005777 (Peroxisome), GO:0005840 (Ribosome), GO:0009941 (Chloroplast envelope), GO:0009570 (Chloroplast stroma) and GO:0009534 (Chloroplast thylakoid).(n) Primers used for genotyping.
- https://cdn.elifesciences.org/articles/86030/elife-86030-supp1-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/86030/elife-86030-mdarchecklist1-v2.docx
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Source data 1
Supplementary Data: Data used for all graphs presented in this study.
- https://cdn.elifesciences.org/articles/86030/elife-86030-data1-v2.xlsx
