The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: The Non-Photochemical Quenching (NPQ) protects photosystems from energy overloading by excess light exposure. The NPQ consists of multiple factors which function in different time scales and energy levels. One of the factors, qH, has been proposed based on chlorophyll fluorescence lifetime observation and the plastid lipocalin has been identified as the important player to regulate qH. It remains to link the qH phenotype and molecular mechanisms. The authors purify photosynthetic protein complexes from the qH mutants and tried to build a biophysical model to link qH phenomena and …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: The Non-Photochemical Quenching (NPQ) protects photosystems from energy overloading by excess light exposure. The NPQ consists of multiple factors which function in different time scales and energy levels. One of the factors, qH, has been proposed based on chlorophyll fluorescence lifetime observation and the plastid lipocalin has been identified as the important player to regulate qH. It remains to link the qH phenotype and molecular mechanisms. The authors purify photosynthetic protein complexes from the qH mutants and tried to build a biophysical model to link qH phenomena and protein science based on chlorophyll fluorescence lifetime observation.
Response: Thank you for your constructive comments which we have addressed and complete the manuscript nicely.
Major comments: There are two major issues. One issue is, even many kinetics are presented, but the relationship between these values and qH phenotypes is not clearly stated or connected. One idea is to build the mathematical model(s) to explain these kinetics. The other issue is, lipid composition is not considered. Indeed, this phenomenon is observed or emphasized in low temperatures. Generally thinking, lipid composition bound to photosynthetic complexes would be disturbed or modified its conformation.
Response: ____We have not attempted to build a descriptive model of how the different molecular players in qH operate in the membrane to yield the observed fluorescence kinetics as this is beyond the scope of our study. However, we agree that lipid composition should be considered and we have now added two additional authors, text in the method and result sections and new Fig. 6 and Fig. S9 examining lipid composition of thylakoid extract, LHCII trimer and LHCII/Lhcb monomer fractions. No significant differences can be observed between the qH ON and OFF states in the main chloroplastic lipids.
Minor comments: Some datasets are less biological replicates or not clearly stated about the biological replicate number (Figure 2, Figure 4, Figure 5, Figure S2, Figure S5, and Figure S8). Normally, at least three independent biological replicates are required. Technical replicates are not acceptable.
Response: ____If by biological replicates, you mean three independent plant individuals, we agree that this would be the bare minimum required, and we apologize for the confusion. The definition of biological replicate (also referred to as biological experiment) in our study is each one represents a separate batch of several plant individuals pooled (n = 2 to 8) grown at independent times. Then within each biological experiment, we perform technical replicates (i.e. independent measurements of different aliquots from the same sample) which we believe are acceptable and necessary but we agree not sufficient. For most data, we have at least 2 biological experiments, and up to 3, for assessing the quenched nature of LHCII trimer and not LHCII/Lhcb monomer (Fig. 3). We have rephrased the text so this aspect is clearer and also provide more detail below about the aforementioned figures.
Fig. 2: TCSPC on thylakoids, n=3 technical replicates from 2 independent biological experiments; Two separate thylakoid preparations were made from independently grown plants (leaves from n > or = 5 plants were pooled each time). Fig. 4: CN-PAGE, n=3 technical replicates from 2 independent biological experiments (leaves from n > or = 3 plants were pooled each time). Fig. 5: TCSPC on isolated complexes, n=3 technical replicates from 2 independent biological experiments; Two separate thylakoid preparations were made from independently grown plants (leaves from n = 8 plants were pooled each time). Fig. S2: step solubilization, n=2 technical replicates; here 1 biological experiment was used from n = 8 plants. Fig. S5 contains the biological replicates 1 (n=2 plants) and 2 (n=8 plants) of the representative experiment shown in Fig3, biological replicate 3 (n=8 plants). Fig. S8: HPLC on isolated complexes from 2 independent biological experiments (leaves from n = 8 plants were pooled each time).
In Figure 3 and Figure S3, extend the length of the major tick for each axis. It is hard to distinguish between major tick and minor tick.
Response: Ok, done.
In Figure 3, mark the measured peak wavelength value on the top for readers.
Response: ____Ok, done, added in the legend “with maxima at 679 nm for all samples”.
In Figure 4, Why do not you present chlorophyll kinetics? I suspect it is possible to acquire if you used SpeedZen.
Response: In Figure 4, we present a measurement of fluorescence emission from separated pigment-protein complexes by CN-PAGE, there are no light-induced changes to be measured here hence we do not present chlorophyll fluorescence kinetics.
In Figure 6, decrease the thickness of the border for the bar graph or marker. Markers on the top of the bar graph are not visible.
Response: Ok, done.
Figures S3 and S6, provide the elution volume of protein standard in the chromatograph.
Response: We don’t make any statement regarding the molecular weights of the protein complexes from the chromatograms, so elution volumes of protein standards are not required. Composition of the different peaks were validated by Iwai et al. 2015 (Nat Plants 1: 14008) and further verified here (Fig. S6, S10).
Figures S11 and S12, describe the number of biological replicates.
Response: ____Ok, done (now Fig. S12 and S13).
Reviewer #1 (Significance (Required)): The topic is important for plant physiology especially photosynthesis regulation and biophysical characterization is straightforward to interpret molecular machinery. Other studies are only for chlorophyll observation for the whole plant body, but most importantly, this study is the challenging work on qH characterization with a biochemical approach.
Response: Thank you for your appreciation of our work!
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Authors observed qH in isolated LHCII trimers with Chl fluorescence changes (shorter), and concluded that no single major Lhcb isomers is necessary for qH.
Response: Thank you for your constructive comments which we have now addressed and make the manuscript clearer.
Major concern is: LHCII trimers are divided into S, M, L trimers with different compositions. Authors are requested to interpret their results in terms of L-, M-, S-trimers.
Response: Our solubilization conditions and isolation method don’t allow to distinguish between loosely (L), moderately (M) or strongly (S)-bound trimers, the LHCII trimer fraction is a pool of these trimers. We have now specified this aspect in the discussion and cannot interpret our results further than narrowing down the LHCII trimer as a quenching site. In future work, we will attempt their separation although getting entirely pure fractions of each is technically challenging.
Minor comments are: Authors describes qI as reversible NPQ, but qI with D1 damage is not reversible.
Response: ____D1 can be repaired thereby relaxing qI, see recent article from Nawrocki et al. Sci Adv 2021. We have clarified this point in the introduction.
In page 3 - 2nd paragraph, Authors define components of NPQ one by one, but the definition or recovery kinetics for qH is skipped, And authors suddenly start explaining molecular players of qH without changing paragraph.
Response: We have now clarified that the relaxation kinetics for sustained NPQ including qH are slow (hours to days) and changed paragraph to introduce the molecular players known to be regulating qH.
In Fig. S6, authors tried to confirm the trimer and monomer fractions they used by using Lhcb2 and Lhcb4 antibodies, respectively. But, the distribution of Lhcb2 only in Trimer fraction in WT, which is different from the distribution in other mutants. Contamination of Lhcb4 in Trimer fraction is also of concern. Authors may use BN-PAGE or Ultracentrifugal separation, rather than gel filtration.
Response: Regarding the different distribution of Lhcb2 between WT and mutants, we have now better labeled Fig. S6B so it is clear that WT is non-treated (non-stress condition) and the mutants underwent a cold and high light-treatment (stress condition). This difference may thus be explained by the trimers stability/propensity to be solubilized by the detergent varying between non-stress and stress conditions. It is not a concern as we’re not comparing mutants to WT. Contamination by Lhcb4 in the trimer fraction is neither a concern as its amount is similarly low between the compared samples: soq1 mutant cold HL (qH ON) and soq1 lcnp mutant cold HL (qH OFF). So presence of Lhcb4 cannot account for the observed difference in fluorescence quenching as its quantity does not differ between the ON and OFF states. Importantly, the monomeric fraction, enriched in Lhcb4, does not show fluorescence quenching. We have used CN-PAGE as a complementary approach that showed that LHCII trimers are quenched after a cold and high light-treatment in both WT and soq1 mutants (Fig. 4). These aspects are described page 7 in the results section “qH is observed in isolated major LHCII”. Here we chose not to use BN/CN-PAGE or sucrose gradient ultracentrifugation for the isolation of the trimeric and monomeric fractions for two reasons: they would not be as suitable for TCSPC experiments due to their acrylamide or sucrose content and they would take more time; gel filtration was preferred to limit buffer exchange and time required from plant protein extraction to measurement.
Reviewer #2 (Significance (Required)):
Localization of qH in LHCII trimers is interesting, but not surprizing.
So, authors are recommended to rewrite the significance of their findings.
Response:____ In the last paragraph of the introduction, we have now clarified that this study identifies qH quenching in the LHCII trimers but not in the minor monomeric Lhcbs. Prior to this work, the peripheral antenna as a whole was known to be required for qH, now this study identified the major trimeric LHCII as a quenching site. The novelty and significance of this work is further substantified by the isolation of quenched antenna directly from plants in physiological conditions, as opposed to artificial induction in vitro. Regarding the “surprising” nature of findings in general, please see answer below to reviewer #3.
My expertise: I am working on the movement of L and M trimers in plants under photoinhibitory illumination.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
The studies reported in this manuscript were designed to test the hypothesis that LCNP binds (or modifies) a molecule in the vicinity of (or within) the antenna proteins, under stress conditions. This in turn triggers a conformational change that converts antenna proteins from a light-harvesting to a dissipative state. Experiments were performed to locate the qH quenching site within the peripheral antenna of PSII and determine its sensitivity to Lhcb subunit composition. The authors were able to isolate antenna complexes with active qH that remained quenched after purification. Analysis of these complexes revealed that qH can occur in the major trimeric LHCII complexes. The elegant studies reported in this manuscript have made good use of appropriate molecular techniques and genetic resources. Genome editing and genetic crosses were used to demonstrate that qH is not restricted to inherent regulation of a specific major Lhcb subunit. The data are clearly presented and the data are convincing.
Response: Thank you very much for your appreciation of our work!
Reviewer #3 (Significance (Required)):
The studies reported in this manuscript build on a firm foundation of previous work by these authors and others. The conclusions are based on the analysis of Chl fluorescence lifetimes in intact leaves, thylakoids, and isolated antenna complexes in which qH was "ON" or "OFF". The findings are interesting and incremental in terms of increasing current understanding. However, the data extend our knowledge of the location of qH within the peripheral antenna of PSII. Rather unsurprisingly, the authors highlight the need to preserve thylakoid membrane macroorganisation for a full qH response.
Response: The philosophical concept of findings not being surprising could be discussed at length. To quote a commenter from this blog: ____https://blogs.uw.edu/ajko/2009/09/17/whats-surprising/____, just because one could have guessed the outcome of an experiment is not the same as empirically validating it. We hope you agree. Plus, as Fabrice Rappaport used to say, “we’re never sheltered from a discovery” and it could have been that isolated LHCII with qH ON showed short Chl fluorescence lifetimes similar to observed in leaves. We couldn’t know until we tried!
Data are presented showing that while qH occurs in the trimeric LHCII complexes, it does not require a specific Lhcb subunit and is insensitive to Lhcb composition. However, the discussion is rather speculative because data interpretation is limited by an absence of knowledge regarding what happens to the LHC trimers and qH during isolation of thylakoids and photosynthetic complexes. This point is considered appropriately in the discussion. The authors also acknowledge the existence of additional quenching sites beyond the LHCII trimers that are required for qH.
Response: Indeed, thank you we have addressed these points in the discussion, and have now added new data on the lack of changes in lipid composition in the LHCII trimer with qH ON or OFF. We view this study as an important milestone to obtain knowledge on the molecular origin of qH.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
The studies reported in this manuscript were designed to test the hypothesis that LCNP binds (or modifies) a molecule in the vicinity of (or within) the antenna proteins, under stress conditions. This in turn triggers a conformational change that converts antenna proteins from a light-harvesting to a dissipative state. Experiments were performed to locate the qH quenching site within the peripheral antenna of PSII and determine its sensitivity to Lhcb subunit composition. The authors were able to isolate antenna complexes with active qH that remained quenched after purification. Analysis of these complexes revealed that qH can occur …
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 #3
Evidence, reproducibility and clarity
The studies reported in this manuscript were designed to test the hypothesis that LCNP binds (or modifies) a molecule in the vicinity of (or within) the antenna proteins, under stress conditions. This in turn triggers a conformational change that converts antenna proteins from a light-harvesting to a dissipative state. Experiments were performed to locate the qH quenching site within the peripheral antenna of PSII and determine its sensitivity to Lhcb subunit composition. The authors were able to isolate antenna complexes with active qH that remained quenched after purification. Analysis of these complexes revealed that qH can occur in the major trimeric LHCII complexes. The elegant studies reported in this manuscript have made good use of appropriate molecular techniques and genetic resources. Genome editing and genetic crosses were used to demonstrate that qH is not restricted to inherent regulation of a specific major Lhcb subunit. The data are clearly presented and the data are convincing.
Significance
The studies reported in this manuscript build on a firm foundation of previous work by these authors and others. The conclusions are based on the analysis of Chl fluorescence lifetimes in intact leaves, thylakoids, and isolated antenna complexes in which qH was "ON" or "OFF". The findings are interesting and incremental in terms of increasing current understanding. However, the data extend our knowledge of the location of qH within the peripheral antenna of PSII. Rather unsurprisingly, the authors highlight the need to preserve thylakoid membrane macroorganisation for a full qH response.
Data are presented showing that while qH occurs in the trimeric LHCII complexes, it does not require a specific Lhcb subunit and is insensitive to Lhcb composition. However, the discussion is rather speculative because data interpretation is limited by an absence of knowledge regarding what happens to the LHC trimers and qH during isolation of thylakoids and photosynthetic complexes. This point is considered appropriately in the discussion. The authors also acknowledge the existence of additional quenching sites beyond the LHCII trimers that are required for qH.
-
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
Authors observed qH in isolated LHCII trimers with Chl fluorescence changes (shorter), and concluded that no single major Lhcb isomers is necessary for qH.
Major concern is: LHCII trimers are divided into S, M, L trimers with different compositions. Authors are requested to interpret their results in terms of L-, M-, S-trimers.
Minor comments are: Authors describes qI as reversible NPQ, but qI with D1 damage is not reversible.
In page 3 - 2nd paragraph, Authors define components of NPQ one by one, but the definition or revoery kinetics for qH is skipped, And authors suddenly start explaining molecular players of qH without changing …
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
Authors observed qH in isolated LHCII trimers with Chl fluorescence changes (shorter), and concluded that no single major Lhcb isomers is necessary for qH.
Major concern is: LHCII trimers are divided into S, M, L trimers with different compositions. Authors are requested to interpret their results in terms of L-, M-, S-trimers.
Minor comments are: Authors describes qI as reversible NPQ, but qI with D1 damage is not reversible.
In page 3 - 2nd paragraph, Authors define components of NPQ one by one, but the definition or revoery kinetics for qH is skipped, And authors suddenly start explaining molecular players of qH without changing paragraph.
In Fig. S6, authors tried to confirm the trimer and monomer fractions they used by using Lhcb2 and Lhcb4 antibodies, respectively. But, the distribution of Lhcb2 only in Trimer fraction in WT, which is diferetnf from the distribution in other mutants. Contamination of Lhcb4 in Trimer fraction is also of concern. Authors may use Bn-PAGE or Ultracentrigugal separation, rather than gel filtration.
provide evidences for
Significance
Localization of qH in LHCII trimers is interesting, but not surprizing.
So, authors are recommended to rewrite the significance of their findings.
My expertise: I am working on the movement of L and M trimers in plants under photoinhibitory illumination.
-
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
Summary:
The Non-Photochemical Quenching (NPQ) protects photosystems from energy overloading by excess light exposure. The NPQ consists of multiple factors which function in different time scales and energy levels. One of the factors, qH, has been proposed based on chlorophyll fluorescence lifetime observation and the plastid lipocalin has been identified as the important player to regulate qH. It remains to link the qH phenotype and molecular mechanisms. The authors purify photosynthetic protein complexes from the qH mutants and tried to build a biophysical model to link qH phenomena and protein science based on chlorophyll …
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
Summary:
The Non-Photochemical Quenching (NPQ) protects photosystems from energy overloading by excess light exposure. The NPQ consists of multiple factors which function in different time scales and energy levels. One of the factors, qH, has been proposed based on chlorophyll fluorescence lifetime observation and the plastid lipocalin has been identified as the important player to regulate qH. It remains to link the qH phenotype and molecular mechanisms. The authors purify photosynthetic protein complexes from the qH mutants and tried to build a biophysical model to link qH phenomena and protein science based on chlorophyll fluorescence lifetime observation.
Major comments:
There are two major issues. One issue is, even many kinetics are presented, but the relationship between these values and qH phenotypes is not clearly stated or connected. One idea is to build the mathematical model(s) to explain these kinetics. The other issue is, lipid composition is not considered. Indeed, this phenomenon is observed or emphasized in low temperatures. Generally thinking, lipid composition bound to photosynthetic complexes would be disturbed or modified its conformation.
Minor comments:
Some datasets are less biological replicates or not clearly stated about the biological replicate number (Figure 2, Figure 4, Figure 5, Figure S2, Figure S5, and Figure S8). Normally, at least three independent biological replicates are required. Technical replicates are not acceptable.
In Figure 3 and Figure S3, extend the length of the major tick for each axis. It is hard to distinguish between major tick and minor tick.
In Figure 3, mark the measured peak wavelength value on the top for readers.
In Figure 4, Why do not you present chlorophyll kinetics? I suspect it is possible to acquire if you used SpeedZen. In Figure 6, decrease the thickness of the border for the bar graph or marker. Markers on the top of the bar graph are not visible.
Figures S3 and S6, provide the elution volume of protein standard in the chromatograph.
Figures S11 and S12, describe the number of biological replicates.
Significance
The topic is important for plant physiology especially photosynthesis regulation and biophysical characterization is straightforward to interpret molecular machinery. Other studies are only for chlorophyll observation for the whole plant body, but most importantly, this study is the challenging work on qH characterization with a biochemical approach.
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