The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts
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eLife assessment
In this important study, the role of NBR1 in the degradation of photodamaged chloroplasts is analyzed, advancing our knowledge of chloroplast homeostasis in response to environmental stress. The evidence presented is convincing, in some parts even compelling, and the results are valuable for the plant and the autophagy research community.
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Abstract
The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Lee and colleagues address the participation of NBR1 in chloroplast clearance after treatment with high light intensity. Authors use NBR1 fused to reporter proteins (GFP, mCherry), with the aid of nbr1, atg7, and nbr1-atg7 mutants, in combination with immunogold labelling to show localization of NBR1 to surface and interior of photodamaged chloroplasts, which follows with their engulfment in the vacuole, a process which is independent of ATG7. The combined use of ATG8 fused to GFP further shows that NBR1 and ATG8 are recruited independently to photodamaged chloroplasts. In addition, the use of mutant versions of NBR1 in combination with mutants lacking E3 ligases PUB4 and SP1 and mutant toc132-2 and tic40-4 lacking members of the TIC-TOC complex of protein translocation …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Lee and colleagues address the participation of NBR1 in chloroplast clearance after treatment with high light intensity. Authors use NBR1 fused to reporter proteins (GFP, mCherry), with the aid of nbr1, atg7, and nbr1-atg7 mutants, in combination with immunogold labelling to show localization of NBR1 to surface and interior of photodamaged chloroplasts, which follows with their engulfment in the vacuole, a process which is independent of ATG7. The combined use of ATG8 fused to GFP further shows that NBR1 and ATG8 are recruited independently to photodamaged chloroplasts. In addition, the use of mutant versions of NBR1 in combination with mutants lacking E3 ligases PUB4 and SP1 and mutant toc132-2 and tic40-4 lacking members of the TIC-TOC complex of protein translocation to the chloroplast, authors show that chloroplast localization of NBR1 requires the ubiquitin ligase domain (UBA2) of the protein, whereas, the PB1 domain exerts a negative effect on NBR1 chloroplast association, yet neither the PUB4 and SP1 E3 ligases nor the TOC-TIC are required for NBR1 association to photodamaged chloroplasts. All these approaches are well described and strongly support the authors' conclusions that the loss of chloroplast envelope integrity allows the entrance of cytosolic ubiquitin ligases and the participation of NBR1 in photodamaged chloroplast clearance by a process of microautophagy. All these findings add valuable information to our knowledge of chloroplast homeostasis in response to light stress.
To further support these conclusions, authors perform a chloroplast proteomic analysis of the WT, nbr1, atg7, and nbr1-atg7 mutants. However, in contrast with the above results, the description of the proteomic data is rather confusing. The paragraph on Page 17 (lines 393-406) is hard to follow. The term "over-representation of less abundant chloroplast protein" is also quite confusing, like the data in Fig. 6 and supplementary to this figure (what does show the PCA analysis in Fig. 6-suppl. 1?). I wonder whether it would be possible to show all these data as supplementary and try to present the data supporting the major conclusion of these analyses (if I understood correctly, that nbr1, atg7, and the double mutant have lower contents of chloroplast proteins), in a more simple and clear format.
Following the reviewer’s comments, we have re-written the result section describing the proteomic data to make it more concise and clearer. We have also made modified Figure 6 to make it more concise and generated new graphs for Figure 6 supplemental figures 1 and 2.
Reviewer #2 (Public Review):
The authors conducted a wide-ranging series of experiments which lead to the conclusion that NBR1 is involved in the clearance of photodamaged chloroplasts. It is a novel finding because the role of NBR1 in this process was never documented. Notably, the NBR1-mediated clearance is only one of the several possible mechanisms responsible for chloroplast turnover. It is not surprising, considering that the nbr1 mutants are viable. The work is arranged very well. The rationale of the subsequent experiments is logically justified and the outcomes and followed by clear conclusions. In consequence, the authors managed not only to observe the association of NBR1 with the chloroplasts but they threw some light on the corresponding mechanisms. The manuscript contains numerous high-quality images from a confocal microscope and from a transmission electron microscope. All images are accompanied by statistical analysis of the respective microscopic observations, which greatly improves the credibility of the conclusions. Shortly, the authors demonstrated that NBR1 decorates not only the exterior but also the interior of damaged chloroplasts in an ATG7-independent way. Next, they establish that NBR1 and ATG8 are recruited to different populations of damaged chloroplasts, and they document differences in chloroplasts turnover, differences in chlorophyll abundance and chlorophyll photochemical properties, as well as differences in the total proteome of the nbr1 mutant in comparison to the wild type and atg7 mutant in two light regimes (low light and high light). Finally, they exclude the requirement for the known E3 ligases PUB4 and SP1 for NBR1mediated degradation and show that the NBR1 internalization relies rather on the chloroplastic membrane rupture than on the TIC-TOC-dependent processes. In summary, the authors postulate that NBR1-mediated chloroplast clearance is a novel, not yet described mechanism and summarize it in a clear diagram.
The work is interesting, the figures are convincing and the conclusions are justified by the results. It provides novel data on the function of selective autophagy receptors NBR1 in plant cells, however, it also leaves the reader with some unanswered questions. The most important is the relative contribution of each of the chloroplast's degradation routes to the turnover of these organelles in different stresses, light regimes, plant growth stages, etc. This is a difficult problem because the mutations in relevant genes have pleiotropic effects and it is difficult to separate the functions of the individual turnover routes. For example, the defects in core autophagy genes (like the atg7 mutant used in this study) result in an increased level of NBR1. These issues are not sufficiently addressed in the discussion.
The reviewer is correct and indeed, we also detected higher levels of NBR1 in the atg7 mutant (Fig 2G). This could be, for example, the underlying reason why there are more chloroplasts decorated with NBR1 in that atg7 mutants than in complemented nbr1 plants, 24h after high light treatment (Fig 1F). However, the higher frequency of photodamaged chloroplasts observed in atg7 (Fig 2D), supports a different scenario: the higher number of photodamaged chloroplasts that are not successfully repaired or degraded by canonical autophagy in atg7, become substrates of NBR1. The increased levels of NBR1 in the agt7 mutant and how this could influence the effects seen in the mutants studied in this manuscript is now discussed in lines 670-673.
Reviewer #3 (Public Review):
The authors use an impressive array of techniques to determine the role of the NBR1 autophagy receptor protein specifically in the clearing of photodamaged chloroplasts. The authors describe the mechanism(s) by which this receptor operates in this context and demonstrate that this NBR1-mediated process occurs independently of SP1 and PUB4 (whose own roles in other aspects of chloroplast autophagy have previously been shown). The authors further dissect the functional domains of NBR1 to identify which are important in this process.
The major strength of this work is the myriad techniques used to approach the problem. The data are of high quality, and on the whole, well replicated and statistically analysed. In the main, these data substantiate the findings of the authors, although some findings are quite correlative/descriptive. However, the authors show good circumspection in their conclusions and discussion. One potential weakness is that the genetic data (use of mutants) rely on single mutant alleles, therefore whilst genetic linkage to the mutations is assumed, it cannot strictly be guaranteed. The authors performed effective genetic complementation to analyse the domain structure of NBR1 shown in Figure 7. It would have been good if complementation of nbr1 and atg1 mutants and/or alternative mutant alleles had been used for experiments described in Figures 1 to 6. Without this, I think even more circumspection regarding the data obtained from these single-allele mutants would be advised.
We agree with the reviewer that more mutant alleles would have provided stronger support to our conclusions, but we would also like to highlight that the atg7-2 (Chung et al 2010), nbr1-2, and atg7-2 nbr1-2 mutants (Jung et al 2020) have been well characterized previously and the nbr1-2 mutant, shown to be rescued by the expression of fluorescently tagged NBR1 (Jung et al 2020). We are confident about the results on the localization of NBR1 in chloroplasts, not only because the fluorescently tagged NBR1 proteins are functional but also because we were able to corroborate the localization of NBR1 by using antibodies against the native proteins (Fig 2). That said, the reviewer does raise an important point and therefore, we have acknowledged more explicitly the limitation of our conclusions based on the analysis of single mutant alleles in lines 630-631 of the discussion.
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eLife assessment
In this important study, the role of NBR1 in the degradation of photodamaged chloroplasts is analyzed, advancing our knowledge of chloroplast homeostasis in response to environmental stress. The evidence presented is convincing, in some parts even compelling, and the results are valuable for the plant and the autophagy research community.
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Reviewer #1 (Public Review):
In this manuscript, Lee and colleagues address the participation of NBR1 in chloroplast clearance after treatment with high light intensity. Authors use NBR1 fused to reporter proteins (GFP, mCherry), with the aid of nbr1, atg7, and nbr1-atg7 mutants, in combination with immunogold labelling to show localization of NBR1 to surface and interior of photodamaged chloroplasts, which follows with their engulfment in the vacuole, a process which is independent of ATG7. The combined use of ATG8 fused to GFP further shows that NBR1 and ATG8 are recruited independently to photodamaged chloroplasts. In addition, the use of mutant versions of NBR1 in combination with mutants lacking E3 ligases PUB4 and SP1 and mutant toc132-2 and tic40-4 lacking members of the TIC-TOC complex of protein translocation to the …
Reviewer #1 (Public Review):
In this manuscript, Lee and colleagues address the participation of NBR1 in chloroplast clearance after treatment with high light intensity. Authors use NBR1 fused to reporter proteins (GFP, mCherry), with the aid of nbr1, atg7, and nbr1-atg7 mutants, in combination with immunogold labelling to show localization of NBR1 to surface and interior of photodamaged chloroplasts, which follows with their engulfment in the vacuole, a process which is independent of ATG7. The combined use of ATG8 fused to GFP further shows that NBR1 and ATG8 are recruited independently to photodamaged chloroplasts. In addition, the use of mutant versions of NBR1 in combination with mutants lacking E3 ligases PUB4 and SP1 and mutant toc132-2 and tic40-4 lacking members of the TIC-TOC complex of protein translocation to the chloroplast, authors show that chloroplast localization of NBR1 requires the ubiquitin ligase domain (UBA2) of the protein, whereas, the PB1 domain exerts a negative effect on NBR1 chloroplast association, yet neither the PUB4 and SP1 E3 ligases nor the TOC-TIC are required for NBR1 association to photodamaged chloroplasts. All these approaches are well described and strongly support the authors' conclusions that the loss of chloroplast envelope integrity allows the entrance of cytosolic ubiquitin ligases and the participation of NBR1 in photodamaged chloroplast clearance by a process of microautophagy. All these findings add valuable information to our knowledge of chloroplast homeostasis in response to light stress.
To further support these conclusions, authors perform a chloroplast proteomic analysis of the WT, nbr1, atg7, and nbr1-atg7 mutants. However, in contrast with the above results, the description of the proteomic data is rather confusing. The paragraph on Page 17 (lines 393-406) is hard to follow. The term "over-representation of less abundant chloroplast protein" is also quite confusing, like the data in Fig. 6 and supplementary to this figure (what does show the PCA analysis in Fig. 6-suppl. 1?). I wonder whether it would be possible to show all these data as supplementary and try to present the data supporting the major conclusion of these analyses (if I understood correctly, that nbr1, atg7, and the double mutant have lower contents of chloroplast proteins), in a more simple and clear format.
-
Reviewer #2 (Public Review):
The authors conducted a wide-ranging series of experiments which lead to the conclusion that NBR1 is involved in the clearance of photodamaged chloroplasts. It is a novel finding because the role of NBR1 in this process was never documented. Notably, the NBR1-mediated clearance is only one of the several possible mechanisms responsible for chloroplast turnover. It is not surprising, considering that the nbr1 mutants are viable. The work is arranged very well. The rationale of the subsequent experiments is logically justified and the outcomes and followed by clear conclusions. In consequence, the authors managed not only to observe the association of NBR1 with the chloroplasts but they threw some light on the corresponding mechanisms. The manuscript contains numerous high-quality images from a confocal …
Reviewer #2 (Public Review):
The authors conducted a wide-ranging series of experiments which lead to the conclusion that NBR1 is involved in the clearance of photodamaged chloroplasts. It is a novel finding because the role of NBR1 in this process was never documented. Notably, the NBR1-mediated clearance is only one of the several possible mechanisms responsible for chloroplast turnover. It is not surprising, considering that the nbr1 mutants are viable. The work is arranged very well. The rationale of the subsequent experiments is logically justified and the outcomes and followed by clear conclusions. In consequence, the authors managed not only to observe the association of NBR1 with the chloroplasts but they threw some light on the corresponding mechanisms. The manuscript contains numerous high-quality images from a confocal microscope and from a transmission electron microscope. All images are accompanied by statistical analysis of the respective microscopic observations, which greatly improves the credibility of the conclusions. Shortly, the authors demonstrated that NBR1 decorates not only the exterior but also the interior of damaged chloroplasts in an ATG7-independent way. Next, they establish that NBR1 and ATG8 are recruited to different populations of damaged chloroplasts, and they document differences in chloroplasts turnover, differences in chlorophyll abundance and chlorophyll photochemical properties, as well as differences in the total proteome of the nbr1 mutant in comparison to the wild type and atg7 mutant in two light regimes (low light and high light). Finally, they exclude the requirement for the known E3 ligases PUB4 and SP1 for NBR1-mediated degradation and show that the NBR1 internalization relies rather on the chloroplastic membrane rupture than on the TIC-TOC-dependent processes. In summary, the authors postulate that NBR1-mediated chloroplast clearance is a novel, not yet described mechanism and summarize it in a clear diagram.
The work is interesting, the figures are convincing and the conclusions are justified by the results. It provides novel data on the function of selective autophagy receptors NBR1 in plant cells, however, it also leaves the reader with some unanswered questions. The most important is the relative contribution of each of the chloroplast's degradation routes to the turnover of these organelles in different stresses, light regimes, plant growth stages, etc. This is a difficult problem because the mutations in relevant genes have pleiotropic effects and it is difficult to separate the functions of the individual turnover routes. For example, the defects in core autophagy genes (like the atg7 mutant used in this study) result in an increased level of NBR1. These issues are not sufficiently addressed in the discussion.
-
Reviewer #3 (Public Review):
The authors use an impressive array of techniques to determine the role of the NBR1 autophagy receptor protein specifically in the clearing of photodamaged chloroplasts. The authors describe the mechanism(s) by which this receptor operates in this context and demonstrate that this NBR1-mediated process occurs independently of SP1 and PUB4 (whose own roles in other aspects of chloroplast autophagy have previously been shown). The authors further dissect the functional domains of NBR1 to identify which are important in this process.
The major strength of this work is the myriad techniques used to approach the problem. The data are of high quality, and on the whole, well replicated and statistically analysed. In the main, these data substantiate the findings of the authors, although some findings are quite …
Reviewer #3 (Public Review):
The authors use an impressive array of techniques to determine the role of the NBR1 autophagy receptor protein specifically in the clearing of photodamaged chloroplasts. The authors describe the mechanism(s) by which this receptor operates in this context and demonstrate that this NBR1-mediated process occurs independently of SP1 and PUB4 (whose own roles in other aspects of chloroplast autophagy have previously been shown). The authors further dissect the functional domains of NBR1 to identify which are important in this process.
The major strength of this work is the myriad techniques used to approach the problem. The data are of high quality, and on the whole, well replicated and statistically analysed. In the main, these data substantiate the findings of the authors, although some findings are quite correlative/descriptive. However, the authors show good circumspection in their conclusions and discussion. One potential weakness is that the genetic data (use of mutants) rely on single mutant alleles, therefore whilst genetic linkage to the mutations is assumed, it cannot strictly be guaranteed. The authors performed effective genetic complementation to analyse the domain structure of NBR1 shown in Figure 7. It would have been good if complementation of nbr1 and atg1 mutants and/or alternative mutant alleles had been used for experiments described in Figures 1 to 6. Without this, I think even more circumspection regarding the data obtained from these single-allele mutants would be advised.
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