Lipid bilayer properties potentially contributed to the evolutionary disappearance of betaine lipids in seed plants

  1. Stéphanie Bolik
  2. Alexander Schlaich
  3. Tetiana Mukhina
  4. Alberto Amato
  5. Olivier Bastien
  6. Emanuel Schneck
  7. Bruno Demé
  8. Juliette Jouhet

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Abstract

Background

Many organisms rely on mineral nutrients taken directly from the soil or aquatic environment, and therefore, developed mechanisms to cope with the limitation of a given essential nutrient. For example, photosynthetic cells have well-defined responses to phosphate limitation, including the replacement of cellular membrane phospholipids with non-phosphorous lipids. Under phosphate starvation, phospholipids in extraplastidial membranes are replaced by betaine lipids in microalgae. In higher plants, the synthesis of betaine lipid is lost, driving plants to other strategies to cope with phosphate starvation where they replace their phospholipids by glycolipids.

Results

The aim of this work was to evaluate to what extent betaine lipids and PC lipids share physicochemical properties and could substitute for each other. By neutron diffraction experiments and dynamic molecular simulation of two synthetic lipids, the dipalmitoylphosphatidylcholine (DPPC) and the dipalmitoyl-diacylglyceryl-N,N,N-trimethylhomoserine (DP-DGTS), we found that DP-DGTS bilayers are thicker than DPPC bilayers and therefore are more rigid. Furthermore, DP-DGTS bilayers are more repulsive, especially at long range, maybe due to unexpected unscreened electrostatic contribution. Finally, DP-DGTS bilayers could coexist in the gel and fluid phases.

Conclusion

The different properties and hydration responses of PC and DGTS provide an explanation for the diversity of betaine lipids observed in marine organisms and for their disappearance in seed plants.

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  1. Version published to 10.1186/s12915-023-01775-z
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    1. General Statements

    We thank the reviewers for their positive statement and the significance of our work.

    2. Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This paper contains a set of highly valuable information on the physicochemical parameteters of betain lipids - which are synthesized in microalgae and some other lower eukaryotic organisms.

    The authors, using advanced biophysical techniques - neutron diffraction and small-angle scattering (SANS) as well as molecular dynamics (MD) simulations - established key physicochemical parameters of synthetic betaine lipid DP-DGTS, and compared it with those of the DPPC phospholipid. They "show that DP-DGTS bilayers are thicker, more rigid, and mutually more repulsive than DPPC bilayers". These are important findings.

    The authors also analyzed the phylogenetic tree of the appearance and disappearance of DGTS biosynthesis enzymes, which - together with the observed "different properties and hydration response of PC and DGTS" led them to explain "the diversity of betaine lipids observed in marine organisms and for their disappearance in seed plants". The authors tentatively suggest "A physicochemical cause of betaine lipid evolutionary loss in seed plants" (Title with "?")

    We put a question mark because our work suggests that the difference of sensitivity to hydration between DGTS and PC bilayers could be an explanation for the betaine lipid disappearance in seed plants due to the dry stage of the seed. In our hands, we never managed to obtain 35S-BTA1 overexpressing plant that produce seed. However, we do not have a formal evidence for this fact. We propose to change the title into: “The possible role of lipid bilayer properties in the evolutionary disappearance of betaine lipids in seed plants.”

    May major concerns with this suggestion are:

    • In thylakoid membranes (TMs) the only phospholipid, PG, plays key roles in PSII and PSI functions (Wada and Murata 2007 Photosynth Res, Hagio et al. Plant Physiol 2000, Domonkos et al. 2004 Plant Physiol; it is difficult to explain how these roles would be overtaken by betaine lipids. In fact, data of Huang et al. (https://www.sciencedirect.com/science/article/pii/S2211926418309366) indicate betaine lipids constitute the major compounds of non-plastidial membranes" and compensation mechanism operate according to which "by the increase of PG in thylakoid membranes, suggesting a transfer of P from non-plastidial membranes to chloroplasts that would maintain a stable lipid composition of thylakoid membranes".
    • Although neutron diffraction and SANS data, as well as MD simulationa might indicate important differences, the behavior of membranes (e.g. stacking interactions, overall structure and structural dynamics of TMs, protein embedding conditions / membrane thickness etc), TMs are more dominantly determined by protein-protein interactions, mainly because these membranes, contain only small areas occupied by the bilayer phase. Similar arguments hold true for the inner mitochondrial membranes (IMMs). I suggest to take into account these severe limitations when extrapolating the data and trying to reach general conclusions. In general, I suggest a more cautious interpretation of data.

    We fully agree with the reviewer’s comments. We indeed wrote in the introduction: “In algae, under phosphate starvation, a situation commonly met in the environment, betaine lipids replace phospholipids in extraplastidic membranes. Because betaine lipids are localized in these membranes [11, 12] and share a common structural fragment with the main extraplastidic phospholipid phosphatidylcholine (PC) (Figure 1A and B), it can be speculated that these two lipid classes are interchangeable, but this was never demonstrated.”

    Plastidial membranes are mainly composed of the non-phosphorus glycerolipids MGDG, DGDG and SQDG. It is well known that in phosphate starvation, in plants and algae, the main phospholipid present in thylakoid membranes, PG, is replaced by SQDG because they are both anionic and bilayer forming lipids (Hölzl G, Dörmann P. Chloroplast Lipids and Their Biosynthesis. Annu Rev Plant Biol. 2019 Apr 29;70:51-81. doi: 10.1146/annurev-arplant-050718-100202; Endo K, Kobayashi K, Wada H. Sulfoquinovosyldiacylglycerol has an Essential Role in Thermosynechococcus elongatus BP-1 Under Phosphate-Deficient Conditions. Plant Cell Physiol. 2016 Dec;57(12):2461-2471; Van Mooy BA, Rocap G, Fredricks HF, Evans CT, Devol AH. Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments. Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8607-12.; Kobayashi K, Fujii S, Sato M, Toyooka K, Wada H. Specific role of phosphatidylglycerol and functional overlaps with other thylakoid lipids in Arabidopsis chloroplast biogenesis. Plant Cell Rep. 2015 Apr;34(4):631-42.). We recently showed by the same kind of neutron diffraction approaches that PG and SQDG share similar physicochemical properties that can explain their conserved replacement by each other in plastidial membranes (Bolik S, Albrieux C, Schneck E, Demé B, Jouhet J. Sulfoquinovosyldiacylglycerol and phosphatidylglycerol bilayers share biophysical properties and are good mutual substitutes in photosynthetic membranes. Biochim Biophys Acta Biomembr. 2022 Dec 1;1864(12):184037. ). However, nothing is known about mitochondrial membranes and DGTS localization. Because PC is a major lipid component of mitochondria in plants and fungi and PC is absent in Chlamydomonas reinhardtii, mitochondria membranes could contain DGTS at least in Chlamydomonas.

    To clarify this statement, we added in the introduction the sentences: “Betaine lipid synthesis is located in the ER [13,14] and betaine lipids are expected to be absent in photosynthetic membranes [12]. Therefore, this PC-betaine lipid replacement is not expected to occur in photosynthetic membranes. However, it might occur at the surface of the chloroplast envelope where PC might be present [15–17]. Nothing is known about the composition of mitochondrial membranes in algae but because PC is a major lipid component in plant and fungal mitochondria, this replacement might also occur in mitochondria.” In the discussion, we replaced “cellular membrane” with “extraplastidial membrane”.

    A minor point - just to avoid possible misunderstanding: betaine can be present in large quantities in many photosynthetic organisms. A short statement on betaine would help.

    To avoid any confusion with betaine as a soluble molecule and betaine lipid, we added this sentence in the introduction: “The presence of betaine lipids is not linked to the synthesis of betaine, a soluble compound present in almost every organism including most animals, plants, and microorganisms, acting as protectant against osmotic stress [22].”

    **Referee cross-commenting**

    I agree with the evaluation of Reviewer #2 - while keeping mine

    Reviewer #1 (Significance (Required)):

    The physico-chemical properties of betaine lipids have not been established. These lipids - under P starvation of microalgae - accummulate in large quentites. Thus, their detailed characterization and comparison to (otherwise similar) phospolipids are of high importance and advance our knowledge about the roles of these lipids and the organization and structural / functional plasticity of biological membranes.

    As outlined above, I suggest a more cautious interpretation of the data and conclusions regarding e.g. the energy-converting membranes.

    I think the audience is relatively broad: (i) basic research of lipid models and (ii) methodology as well as calling the attention of membrane biologists to the scarcely studied betaine lipids.

    My field is the biophysics photosynthesis - the stability and plasticity of the oxygenic photosynthetic machinery at different levels of complexity; the and closest to this topic is the polymorphic lipid phase behavior of plant TMs.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This manuscript nicely presents the effect of phosphate depletion on how betaine lipids function as effective replacements in a water-rich environment. The mix of computational and wet lab experiments provides details on membrane structure and general effects when phospholipids are changed to betaine lipids. I found this manuscript easy to read and understand and is worthy of publishing. However, I do have a few minor comments below to improve the manuscript.

    Minor Comments:

    Phases in PC lipids with saturated tails: The authors present a gel to liquid crystalline phase change for DPPC at 40oC. However, this is at the ripple-liquid crystalline phase transition and the gel doesn't occur until about 34-35oC. This should be noted in the manuscript.

    We indeed completed the sentence in the first result section by : “The DSC data show a sharp phase transition at 40.2 ± 0.1°C for DPPC corresponding to the transition between the ripple phase and the fluid phase, which is consistent with earlier reports on DPPC large unilamellar vesicles [25].”

    Page 4: I am confused with the following phase: "indicating either weak cooperativity between lipid bilayers or that phase co-existence is not a thermodynamic disadvantage, while this phenomenon is not observed for DPPC bilayers." What is meant by phase co-existence is not a thermodynamic disadvantage? Could this also be due to some frustration in phase coexistence and the presence of a ripple phase that kinetically is inhibited and thus a sharp transition is not observed?

    We did not observe a ripple phase in DP-DGTS as it is defined in DPPC bilayer either by DSC, neutron diffraction or SANS experiments. We don’t know if it exists in DP-DGTS bilayers. What we observe in neutron diffraction is a coexistence of gel phase and fluid phase domains in oriented multilayer films of DP-DGTS over a wide range of humidity whereas for DPPC we observe only a gel phase or a fluid phase. Because the thicknesses of the DP-DGTS bilayers are not so different between the gel phase and the fluid phase, we suppose that the free energy difference between the two phases is very small over a wide osmotic pressure range and that could explain the broad phase transition.

    To further clarify our point, we have reworded the sentence in the following way: “As seen in Figure 2A , by increasing the humidity, DPPC molecules transit from the gel to the fluid phase via a ripple phase through a narrow window of osmotic pressures as previously reported [30,31]. In contrast, DP-DGTS bilayers show a phase coexistence that can be observed over a wide P-range and without the appearance of a third phase that could be attributed to a distinct ripple phase (Figure 2B) before forming a single fluid phase at high humidity (i.e., at low P). Based on DSC and neutron diffraction as two independent techniques, we can safely conclude that the phase transition for DP-DGTS is broad. This observation indicates that the free energy difference between the two phases is very small over a wide osmotic pressure range and may be connected to the shapes of the pressure-distance relations in the two phases, which are discussed further below.” We also added in the legend of figure 4 (SANS experiment): “No ripple phase Pb was detected for DP-DGTS bilayers.”

    DOI for computational methods: The DOI listed computational files (https://doi.org/10.18419/darus-2360) does not work.

    Unfortunately, we did not ask for publication of the URL upon submission of the manuscript and thank the reviewer for carefully checking this. Since DaRUS is a peer-reviewed repository ensuring high quality data sets according to the FAIR principle, peer review is still ongoing. The provided link will work definitely only when the manuscript will be published. In the meantime, we provide a temporary link for reviewing :

    https://darus.uni-stuttgart.de/privateurl.xhtml?token=cbfac341-0e4a-4403-8f73-87bce31ca805

    Reviewer #2 (Significance (Required)):

    This work has broad significance and would be of general interest to those in membrane biophysics to plant biology and evolution. The work nicely touches on all these topics, and I find this fills a gap in details of these betaine lipids structure and relation to evolution in terrestrial vs. marine plants.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript nicely presents the effect of phosphate depletion on how betaine lipids function as effective replacements in a water-rich environment. The mix of computational and wet lab experiments provides details on membrane structure and general effects when phospholipids are changed to betaine lipids. I found this manuscript easy to read and understand and is worthy of publishing. However, I do have a few minor comments below to improve the manuscript.

    Minor Comments:

    1. Phases in PC lipids with saturated tails: The authors present a gel to liquid crystalline phase change for DPPC at 40oC. However, this is at the ripple-liquid crystalline phase transition and the gel doesn't occur until about 34-35oC. This should be noted in the manuscript.
    2. Page 4: I am confused with the following phase: "indicating either weak cooperativity between lipid bilayers or that phase co-existence is not a thermodynamic disadvantage, while this phenomenon is not observed for DPPC bilayers." What is meant by phase co-existence is not a thermodynamic disadvantage? Could this also be due to some frustration in phase coexistence and the presence of a ripple phase that kinetically is inhibited and thus a sharp transition is not observed?
    3. DOI for computational methods: The DOI listed computational files (https://doi.org/10.18419/darus-2360) does not work.

    Significance

    This work has broad significance and would be of general interest to those in membrane biophysics to plant biology and evolution. The work nicely touches on all these topics, and I find this fills a gap in details of these betaine lipids structure and relation to evolution in terrestrial vs. marine plants.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    This paper contains a set of highly valuable information on the physicochemical parameteters of betain lipids - which are synthesized in microalgae and some other lower eukaryotic organisms.

    The authors, using advanced biophysical techniques - neutron diffraction and small-angle scattering (SANS) as well as molecular dynamics (MD) simulations - established key physicochemical parameters of synthetic betaine lipid DP-DGTS, and compared it with those of the DPPC phospholipid. They "show that DP-DGTS bilayers are thicker, more rigid, and mutually more repulsive than DPPC bilayers". These are important findings.

    The authors also analyzed the phylogenetic tree of the appearance and disappearance of DGTS biosynthesis enzymes, which - together with the observed "different properties and hydration response of PC and DGTS" led them to explain "the diversity of betaine lipids observed in marine organisms and for their disappearance in seed plants". The authors tentatively suggest "A physicochemical cause of betaine lipid evolutionary loss in seed plants" (Title with "?")

    May major concerns with this suggestion are:

    • (i) In thylakoid membranes (TMs) the only phospholipid, PG, plays key roles in PSII and PSI functions (Wada and Murata 2007 Photosynth Res, Hagio et al. Plant Physiol 2000, Domonkos et al. 2004 Plant Physiol; it is difficult to explain how these roles would be overtaken by betaine lipids. In fact, data of Huang et al. (https://www.sciencedirect.com/science/article/pii/S2211926418309366) indicate betaine lipids constitute the major compounds of non-plastidial membranes" and compensation mechanism operate according to which "by the increase of PG in thylakoid membranes, suggesting a transfer of P from non-plastidial membranes to chloroplasts that would maintain a stable lipid composition of thylakoid membranes"
    • (ii) Although neutron diffraction and SANS data, as well as MD simulationa might indicate important differences, the behavior of membranes (e.g. stacking interactions, overall structure and structural dynamics of TMs, protein embedding conditions / membrane thickness etc), TMs are more dominantly determined by protein-protein interactions, mainly because these membranes, contain only small areas occupied by the bilayer phase. Similar arguments hold true for the inner mitochondrial membranes (IMMs).

    I suggest to take into account these severe limitations when extrapolating the data and trying to reach general conclusions. In general, I suggest a more cautious interpretation of data.

    A minor point - just to avoid possible misunderstanding: betaine can be present in large quantities in many photosynthetic organisms. A short statement on betaine would help.

    Referee cross-commenting

    I agree with the evaluation of Reviewer #2 - while keeping mine

    Significance

    The physico-chemical properties of betaine lipids have not been established. These lipids - under P starvation of microalgae - accummulate in large quentites. Thus, their detailed characterization and comparison to (otherwise similar) phospolipids are of high importance and advance our knowledge about the roles of these lipids and the organization and structural / functional plasticity of biological membranes.

    As outlined above, I suggest a more cautious interpretation of the data and conclusions regarding e.g. the energy-converting membranes.

    I think the audience is relatively broad: (i) basic research of lipid models and (ii) methodology as well as calling the attention of membrane biologists to the scarcely studied betaine lipids.

    My field is the biophysics photosynthesis - the stability and plasticity of the oxygenic photosynthetic machinery at different levels of complexity; the and closest to this topic is the polymorphic lipid phase behavior of plant TMs.

  5. Version published to 10.1101/2023.01.24.525350v2 on bioRxiv
  6. Version published to 10.1101/2023.01.24.525350v1 on bioRxiv