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

  1. Masanori Izumi
  2. Sakuya Nakamura
  3. Kohei Otomo
  4. Hiroyuki Ishida
  5. Jun Hidema
  6. Tomomi Nemoto
  7. Shinya Hagihara

  • Curated by eLife

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    This manuscript investigates how chloroplasts are broken down during light-limiting conditions as plants reorganize their energy-producing organelles during carbon limitation. The authors provide convincing live-cell imaging data of plastids, documenting that buds form on the surface of chloroplasts and pinch away, then associate with the vacuole via a mechanism that depends on autophagy machinery, but not plastid division machinery. However, the absence of quantitative analyses makes the work incomplete at the current stage. The manuscript nevertheless provides important groundwork for other scientists studying the regulation and breakdown of energy-producing organelles, including chloroplasts and mitochondria.

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Abstract

Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. The buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. These budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast protrusions. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes tightly interact with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B (DRP5B), which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.

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  1. eLife

    eLife assessment

    This manuscript investigates how chloroplasts are broken down during light-limiting conditions as plants reorganize their energy-producing organelles during carbon limitation. The authors provide convincing live-cell imaging data of plastids, documenting that buds form on the surface of chloroplasts and pinch away, then associate with the vacuole via a mechanism that depends on autophagy machinery, but not plastid division machinery. However, the absence of quantitative analyses makes the work incomplete at the current stage. The manuscript nevertheless provides important groundwork for other scientists studying the regulation and breakdown of energy-producing organelles, including chloroplasts and mitochondria.

  2. eLife

    Reviewer #1 (Public Review):

    Summary:
    The authors demonstrated that carbon depletion triggers the autophagy-dependent formation of Rubisco Containing Bodies, which contain chloroplast stroma material, but exclude thylakoids. The authors show that RCBs bud directly from the main body of chloroplasts rather than from stromules and that their formation is not dependent on the chloroplast fission factor DRP5. The authors also observed a transient engulfment of the RBCs by the tonoplast during delivery to the vacuolar lumen.

    Strengths:
    The authors demonstrate that autophagy-related protein 8 (ATG8) co-localizes to the chloroplast demarking the place for RCB budding. The authors provide good-quality time-lapse images and co-localization of the markers corroborating previous observations that RCBs contain only stroma material and do not include thylakoid. The text is very well written and easy to follow.

    Weaknesses:
    A significant portion of the results presented in the study comes across as a corroboration of the previous findings made under different stress conditions: autophagy-dependent formation of RCBs was reported by Ishida et all in 2009. Furthermore, some included results are not of particular relevance to the study's aim. For example, it is unclear what is the importance of the role of SA in the formation of stromules, which do not serve as an origin for the RCBs. Similarly, the significance of the transient engulfment of RCBs by the tonoplast remained elusive. Although it is indeed a curious observation, previously reported for peroxisomes, its presentation should include an adequate discussion maybe suggesting the involved mechanism. Finally, some conclusions are not fully supported by the data: the suggested timing of events poorly aligns between and even within experiments mostly due to high variation and low number of replicates. Most importantly, the discussion does not place the findings of this study into the context of current knowledge on chlorophagy and does not propose the significance of the piece-meal vs complete organelle sequestration into the vacuole under used conditions, and does not dwell on the early localization of ATG8 to the future budding place on the chloroplast.

  3. eLife

    Reviewer #2 (Public Review):

    This manuscript proposed a new link between the formation of chloroplast budding vesicles (Rubisco-containing bodies [RCBs]) and the development of chloroplast-associated autophagosomes. The authors' previous work demonstrated two types of autophagy pathways involved in chloroplast degradation, including piecemeal degradation of partial chloroplast and whole chloroplast degradation. However, the mechanisms underlying piecemeal degradation are largely unknown, particularly regarding the initiation and release of the budding structures. Here, the authors investigated the progression of piecemeal-type chloroplast trafficking by visualizing it with a high-resolution time-lapse microscope. They provide evidence that autophagosome formation is required for the initiation of chloroplast budding, and that stromule formation is not correlated with this process. In addition, the authors also demonstrated that the release of chloroplast-associated autophagosome is independent of a chloroplast division factor, DRP5b.

    Overall, the findings are interesting, and in general, the experiments are very well executed. Although the mechanism of how Rubisco-containing bodies are processed is still unclear, this study suggests that a novel chloroplast division machinery exists to facilitate chloroplast autophagy, which will be valuable to investigate in the future.

  4. eLife

    Reviewer #3 (Public Review):

    Summary:
    Regulated chloroplast breakdown allows plants to modulate these energy-producing organelles, for example during leaf aging, or during changing light conditions. This manuscript investigates how chloroplasts are broken down during light-limiting conditions.

    The authors present very nice time-lapse imaging of multiple proteins as buds form on the surface of chloroplasts and pinch away, then associate with the vacuole. They use mutant analysis and autophagy markers to demonstrate that this process requires the ATG machinery, but not dynamin-related proteins that are required for chloroplast division. The manuscript concludes with a discussion of an internally-consistent model that summarizes the results.

    Strengths:
    The main strength of the manuscript is the high-quality microscopy data. The authors use multiple markers and high-resolution time-lapse imaging to track chloroplast dynamics under light-limiting conditions.

    Weaknesses:
    The main weakness of the manuscript is the lack of quantitative data. Quantification of multiple events is required to support the authors' claims, for example, claims about which parts of the plastid bud, about the dynamics of the events, about the colocalization between ATG8 and the plastid stroma buds, and the dynamics of this association. Without understanding how often these events occur and how frequently events follow the manner observed by the authors (in the 1 or 2 examples presented in each figure) it is difficult to appreciate the significance of these findings.

  5. Version published to 10.1101/2023.10.11.561947v1 on bioRxiv