A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis

  1. Rosa Pipitone
  2. Simona Eicke
  3. Barbara Pfister
  4. Gaetan Glauser
  5. Denis Falconet
  6. Clarisse Uwizeye
  7. Thibaut Pralon
  8. Samuel C Zeeman
  9. Felix Kessler
  10. Emilie Demarsy

  • Curated by eLife

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    Summary: All three reviewers as well as myself are impressed by the in depth and multi-method analysis of chloroplast and thylakoid membrane development provided in your study, including time courses of 3D imaging combining TEM, SBF-SEM and confocal microscopy, lipidomics and proteomics. However, some analyses need to be improved and/or better explained.

    • There is a concern about the proteomics analysis, as the low number of proteins changing in abundance upon de-etiolation is unexpected. It is not clear how the samples were harvested. Were they harvested in the light and could that have influenced protein abundance? The harvesting procedure needs to be better explained. Or is the proteomics method not sensitive enough? The proteomics should be validated, for example by Western Blots with well-established marker proteins such as phyA and HY5.

    • Please also add loading controls to Fig 6 and the associated supplemental figure.

    • Please explain better how the volume of dividing chloroplasts was determined.

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Abstract

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling ‘Structure Establishment Phase’ followed by a ‘Chloroplast Proliferation Phase’ during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.

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  1. Version published to 10.7554/elife.62709 on eLife
  2. eLife

    Reviewer #3:

    This study by Pipitone et al. combines SBF-SEM microscopy with quantitative proteomics and lipidomics to explore chloroplast differentiation. Authors describe that chloroplast biogenesis occurs in a first phase of structure establishment with thylakoid biogenesis, followed by a second phase of chloroplast division. The images and 3D reconstructions are beautiful, the quantitative data are novel, and their integration offers a new perspective into the seedling de-etiolation process, a model system for physiological and molecular studies. However, in my opinion some aspects need to be better explained and significantly improved.

    • In lines 276-282, the authors write: "After 8h of illumination (T8), we observed decreased abundance of only one protein (the photoreceptor cryptochrome 2, consistent with its photolabile property) and increased levels of only three proteins, which belonged to the chlorophyll a/b binding proteins category involved in photoprotection (AT1G44575 = PsbS; AT4G10340= Lhcb5; AT1G15820= Lhcb6". This is striking, as many well studied proteins change in abundance during the first hours of de-etiolation. Actually, looking into the data set with the quantification data for the ~5,000 proteins, it appears that many proteins do show significant changes between T0 and T8. For example PORA and ELIP, changes that are also reflected in figure 6A.

    • Related to the above, well known proteins for example phyA and HY5, that undergo drastic changes in abundance when etiolated seedlings are first exposed to light, do not show changes in T4,T8 and T12 relative to T0 in the proteomics data set. This raises questions about the proteomic approach (sensitivity of the method?) or the experimental setup. Could authors please comment on this? I feel that validation of the proteomics approach is critical, especially taking into account the central conclusion that "the first 12h of illumination saw very few significant changes in protein abundance".

    • Lines 570-572: A reference is needed. Also, it is mentioned that PSII appears later than PSI, which does not seem to match the observation that PSII proteins appear earlier than PSI, or that the surface area occupied at early time points by PSII is greater than the one occupied by PSI. Please check.

    • Are the calculations of thylakoid surface expansion over time consistent with previous available data using tomography? Please include.

    • In the introduction, authors could include mention of the massive transcriptional reprogramming that takes place during de-etiolation. In addition, I think that comparison of the proteomics data with the transcriptomic changes during de-etiolation (well described in the literature) would allow further understanding of the distinct phases proposed. For the chloroplast proteins already present in the dark, how does this correlate with expression of the corresponding genes?

  3. eLife

    Reviewer #2:

    This impressive manuscript describes a comprehensive, multifaceted analysis of the morphological and molecular changes that accompany photosynthetic establishment during seedling de-etiolation. Morphological data, focusing in particular on the photosynthetic thylakoid membranes, are derived using transmission electron microscopy (TEM), serial block face scanning electron microscopy (SBF-SEM), and confocal microscopy, while quantitative molecular data on the abundances of proteins and lipids are derived using mass spectrometry and western blotting. The various data are acquired over a time course between 0 h and 96 h post illumination, and with a high level of temporal resolution. The data allow the authors to develop a mathematical model for the expansion of the surface area of thylakoids (reaching 500-times the surface area of the cotyledon leaf), which matches well with experimental observations from the SBF-SEM analysis for earlier, but not later, stages of de-etiolation. Moreover, the data point to a two-phase organization of the de-etiolation process, with the first phase ("Structure Establishment") characterized by thylakoid assembly and photosynthetic establishment, and the second phase ("Chloroplast Proliferation") characterized by chloroplast division and cell expansion.

    The data are of a high standard, and the depth and breadth of analysis in a single, unified study is unprecedented. While it is arguable that there are few major, completely novel insights reported here (indeed, in the Discussion, the authors very helpfully point out how many of the parameters they have measured are consistent with data reported elsewhere by others), this should not detract from the overall value of the study; a major and unique strength here is that all of the data have been acquired together and so are directly comparable. I have no doubt that this dataset will be extremely interesting to many researchers, and prove to be an invaluable resource for the plant science community. Consequently, I am sure that it will attract many citations.

    I have a few specific comments that I would like the authors to consider carefully, as follows.

    1. Figure 3. The 3D reconstructions are undoubtedly useful for deriving quantitative data, as they enable the derivation of thylakoid surface area data to verify the mathematical model. However, it is very difficult to see anything clearly in the images shown in the Figure. I wonder if the authors can make the images clearer, and then also point to and describe some of the key features. The videos do help a bit, but even these are not that clear.

    2. Page 9, second paragraph. It is here that the "two phases" model is first proposed. I really could not see a clear basis for proposing this model here, using the data that had been presented thus far. As I see it (and based on the way the two phases are described in the Discussion), one can't really propose this model until after the chloroplast number and cell size data have been presented.

    Moreover, the description of the second phase here ("and a second phase...") seems a bit inconsistent with the statement in the paragraph above that thylakoid surface area increases dramatically between T4 and T24, and much less between T24 and T96.

    1. Figure 6, and the related supplementary figure. Loading controls are missing here, and should be added. Also, it is stated that a number of proteins (PsbA, PsbD, PsbO, Lhcb2) are "detectable" at T0 (line 348, page 11). To me, they look UNdetectable.

    2. Dividing chloroplasts. On page 13, line 412-413, it is stated that the volume of dividing chloroplasts was measured, and we are referred to Figures 8E and 4B in support of this statement. However, it is not explained how this was done. More clear and specific explanation is needed. Was it the case that the authors sought out and measured dumbbell-shaped organelles, and quantified those? If so, images are needed to illustrate this point. And, I don't see anything relevant in Fig. 4B - this callout apparently belongs in the following sentence. The statement that the average size of dividing chloroplasts was higher than that of all chloroplasts (lines 413-414) is not really surprising if the authors were measuring organelles just on the point of becoming two organelles.

    3. Page 13, beginning of modelling section. The motivation for this section needs to be better introduced. When I first read it, I could not understand why the authors wished to again "determine the thylakoid membrane surface area", as this had already been discussed earlier in the manuscript.

    Also related to the modelling: Did the authors take into account the existence of appressed membranes when calculating the surface area exposed to the stroma (lines 431-432). And, assuming it is clearly established that there is a 1:1 relationship between these proteins and the relevant complexes (lines 441-443), perhaps this should be stated and the relevant literature cited.

  4. eLife

    Reviewer #1:

    The work by Pipitone et al. is a very carefully performed and technically sophisticated elucidation of the establishment of the thylakoid membrane system in Arabidopsis chloroplasts upon first illumination of cotyledons. Its charm is the three-dimensional resolution during a time course that allows it to follow the rapid changes occuring during the short time window in which the greening occurs. In addition, the authors included proteomics and lipidomics approaches complementing the morphological observations by sound molecular data. All together the study provides a very detailed catalogue of the processes that trigger chloroplast biogenesis that is highly useful for the community as it provides important numbers of size and development.

    Improvements:

    Actually the work has been performed very carefully and there is not much to improve.

    The introduction could contain more references (e.g. lines 77, 83, 90, 93, 98,, 131, 132)

    SBF-SEM should be spelled out at first mentioning (line 146) and maybe a bit more background about the technology would be helpful for the reader to understand it.

    Line 244: The occurrence of starch granules is of course caused by the continuous illumination. It however may also have an impact on the final size of the plastid. It would be interesting to know whether chloroplasts at the end of a night phase are smaller than at the end of a light phase. This is not mandatory for the current manuscript but an interesting question to follow in future and maybe to be discussed.

    Line 251: The surface area.... please define what is meant since membranes have two sides.

    Lines 256-261: There is another study done in cell culture that has a similar design (Dubreuil et al ), are the two studies compatible with each other in their conclusion and if not, what are the differences?

    Lines 549-551: This sentence is not perfectly clear to me. Maybe the authors can explain this a bit more in detail using examples.

    Lines 564-573: I think it is worth noting that the interactions between PSII complexes located in neighbouring thylakoid membranes trigger the stacking of the grana. It is therefore tempting to speculate that stroma lamellae are established first and that these membranes are then stacked after PSII complexes are inserted into the membrane because they provide the adhesion points between them.

  5. eLife

    Summary: All three reviewers as well as myself are impressed by the in depth and multi-method analysis of chloroplast and thylakoid membrane development provided in your study, including time courses of 3D imaging combining TEM, SBF-SEM and confocal microscopy, lipidomics and proteomics. However, some analyses need to be improved and/or better explained.

    • There is a concern about the proteomics analysis, as the low number of proteins changing in abundance upon de-etiolation is unexpected. It is not clear how the samples were harvested. Were they harvested in the light and could that have influenced protein abundance? The harvesting procedure needs to be better explained. Or is the proteomics method not sensitive enough? The proteomics should be validated, for example by Western Blots with well-established marker proteins such as phyA and HY5.

    • Please also add loading controls to Fig 6 and the associated supplemental figure.

    • Please explain better how the volume of dividing chloroplasts was determined.

  6. Version published to 10.1101/2020.08.30.274043v2 on bioRxiv
  7. Version published to 10.1101/2020.08.30.274043v1 on bioRxiv