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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6368548. The manuscript by Cuitun-Coronado et al. investigates the effects of circadian and diel cycles on the photosynthetic processes of the liverwort Marchantia polymorpha. The authors clarified three points: 1) they confirmed that M. polymorpha does display circadian regulation of several photosynthetic parameters; 2) they showed that light-dark cycles mask circadian cycles for these parameters; 3) by using a pharmacological approach, they showed that chloroplast translation is necessary for the circadian regulation of photosynthesis. The study fills a gap of knowledge in the circadian regulation of photosynthetic activity across evolution, by providing an additional link to what was known in cyanobacteria, microalgae, and vascular plants. It is original and well-conducted. The interpretation and the discussion of the results is very convincing, and the conclusions are carefully drawn and backed up by the data. Major comments Page 7, lines 173-176: it is stated that the differences in period length observed between PAM and DF measurements might be due to differences in light intensity. How about light quality, as the PAM measurements were performed under blue light, vs. mixed red/blue for DF? Blue light, which was used in both types of experiments, has been shown to impact circadian rhythms in plants through the action of cryptochromes (e.g. Chaves et al., Annu. Rev. Plant Biol., 2011). Also, BL is responsible for chloroplast relocation responses, which is mediated by phototropins (e.g. see Kong et al. Plant and Cell Physiology, 2013). In Arabidopsis it can also impact plastid transcription through the intermediate of the blue-responsive plastid sigma factor SIG5 (Tsunoyama et al, FEBS Lett. 2002) and it is regulated by BL photoreceptors cryptochromes (Onda 2007, Plant Journal). Can you comment on these potential effects? Has their involvement in Marchantia polymorpha been investigated? Did they motivate your use of blue light for these experiments? Page 13, lines 316-323, please discuss the lack of effect on circadian rhythm by rifampicin: at first it may appear odd that translation has an effect but transcription doesn't. Could it be due to rifampicin only inhibiting PEP and transcription by NEP compensates? Page 14, lines 354-363: Is there a reason why you performed light curves with increasing light intensity up to 789 µmol m-2 s-1 if you end up using only the data with light intensity closer to growth conditions (108 µmol m-2 s-1)? Could the 20 minutes of dark-adaptation every two hours and increasing light intensity up to 789 µmol m-2 s-1 have affected the circadian rhythms of plants that were supposed to be at constant LL? This experimental setup could be considered as fluctuating light condition. Please discuss whether this may have an effect on the observed results. Minor comments Page 2, lines 27-28, "the circadian regulation of several measures of photosynthetic biochemistry (delayed fluorescence, the rate of photosynthetic electron transport, and non-photochemical quenching of chlorophyll fluorescence)": please rephrase this sentence as it seems that the circadian regulation has an effect on the measurements themselves while they are tools to see something happening at photosynthetic levels. We suggest replacing it with "circadian regulation affects photosynthesis performances, detectable by several photosynthetic parameters such as delayed fluorescence, the rate of photosynthetic electron transport, and non-photochemical quenching of chlorophyll fluorescence". Same can be applied in Pages 4-5, lines 98-99. Page 4, lines 84-90, this sentence is very long and difficult to read, please rephrase and split it into at least two sentences for better readability and clarity. Page 5, lines 114-118, discussion might be a better place for this. Page 5, line 118, please replace "Features of PAM fluorescence" with "Features of chlorophyll fluorescence as measured by PAM" as the first indicates a specific method to measure an intrinsic phenomena (Chl fluo) and it is not the only one for Chl fluo measurements. Page 5, line 123, please replace "PAM metrics" with "prompt fluorescence" for consistency with DF, named just before. Page 6, lines 126-127, to be consistent with the nomenclature, "LL" should be "low light", not "constant light". Consider replacing LL with CLL ("constant low light") since the plants are actually exposed to a lower light compared to the standard growth light described in materials and methods. Also, please include in the description of the light conditions "LD" together with its full name (which is directly mentioned before in line 138 without its full name "light-dark cycles"). Pages 6-7, lines 152-154, this sentence is more technical and it does not seem necessary to understand and interpret the results described later. Please consider moving this sentence in Material and methods to keep the results section more readable. Page 7, lines 167-172, please provide a reason why photosynthetic activity was measured only under LL and not also in the other two conditions described for DF. Page 8, lines 196-199, reading the text it seems you used only T24 and T28. Please list all the T lengths (T20, T22, T24 and T28) as reported in Figure 3 legend. Page 10, line 254, please add a short sentence to briefly comment the results obtained with the rifampicin treatment. Page 13, lines 316-318, "Perhaps inhibiting the expression of these proteins prevents or reduces PSII repair, which disrupts circadian cycles of the rate of electron transport by interfering with the electron transport process". Please rephrase this sentence, as it is written it can be interpreted as defective PSII repair disrupts circadian cycles. Page 13, line 335, describe conditions of its natural habitat "such as". Page 14, lines 341, in the growth conditions you describe that the standard light conditions used were 100 μmol photons m-2 s-1 of white light. Is there a reason why for the DF experiment described in this manuscript you decided to use 60 µmol m-2 s-1? Page 18, legend of Figure 1: Line 421, "thallus" should be "thalli"; "light/cycles" should read "light/dark cycles". Line 430, define FFT-NLLS. Please move the definition of RAE from lines 433-434, to its first occurrence line 430. Line 433, define SEM. Page 19, line 470, please write SEM instead of s.e.m., for consistency with the legend of Figure 1. Figure 1: the figure is very big and can create confusion, please consider to split it in two figures, e.g. a-f Figure1 and g-k Figure 2. In the legend, include the abbreviations for the light conditions and correct "ligh/cycles" with "light-dark cycles" as reported in the text. Panel g. Please better describe this panel, especially the colours meaning in the clock. Figure 1 (a,c,e) 2 and Figure 4: the alternance of white and light grey bands to mark the 12h periods can be very easily misinterpreted as light/dark cycles. To clarify and display the constant light conditions at a glance, please consider using another means to evidence them, such as dashed lines. Figure 4: similarly to Figure 1, this figure is very large and hard to read. Consider splitting it. Please write the concentrations of lincomycin and rifampicin in the relevant panels, as the different shades are difficult to distinguish and understand in panels a-d, g-j, m-p and s-v. Panels f-l and r-x: please use the same scale on the y axis, or, if not possible, bring attention of the reader to the different scale in the figure legend. At first, we were puzzled by the 'no-treatment' not starting from similar amplitudes. Maria Paola Puggioni and Aurélie Crepin (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Wolfgang Schröder, Pierrick Bru, Jingfang Hao, Fadime Demirel, Tatyana Shutova, André Graça, Jack Forsman and Domenica Farci.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5884378. The manuscript by Nies et al. demonstrates how changing pulse amplitude modulation (PAM) parameters can affect non-photochemical quenching (NPQ) and photosystem II yield (ՓPSII). Using in silico simulations of PAM experiments, the authors illustrate how NPQ and ՓPSII are affected by varying: i) the delay between measurement of maximal fluorescence Fm and the onset of the actinic light (or between turning off AL and measurement of Fm'' in the dark), ii) the intensity of the actinic light, iii) the frequency of the saturating pulses, iv) and their duration. Nies et al. finish by validating their in silico model, and suggesting that scientists using PAM must provide the details of all of the parameters listed above in the methods section of any publications to allow their experiments to be accurately reproduced and modeled. We enjoyed this manuscript, however, we have some comments and suggestions for improvement, listed below. Major comments - We suggest moving part of the model validation section of the results, shown in Figures 8 (and 9), to the start of the manuscript. This rearrangement would show the reader that the mathematical model used in the in silico simulations can accurately reproduce experimental data, before the parameter-dependent changes to NPQ and ՓPSII are simulated. In the current arrangement, the reader needs to have prior knowledge that the changes in NPQ and ՓPSII shown in the simulations are accurate, before the herein updated model has been validated. - Fig8. Regarding the validation of the mathematical model by comparing to experimental PAM measurements with different SP durations, or different delays of AL onset from Fm measurement, with the simulated data: what is the rationale for choosing these, how about testing the other parameters such as AL intensity and frequency of SP? Please comment on the impact of the different parameters on e.g. the NPQ measurement and rank them by stronger to lower effect based on your simulations and experiments. Also a historical perspective/physiological relevance of delaying the SP from actinic onset would be welcome! How about giving recommendation to researchers in the field to have Fm determination/SP right at onset of illumination, with no delay, to prevent further confusion (and similarly have the final SP in AL on, followed by AL off with no delay). - Line 326. Regarding the use of another model of photosynthesis, we found this very interesting and suggest that a comparison of the simulations generated by the two mathematical models using the same set of parameters be included as a main or supplemental figure, and its description be included in the results section. The GitLab link (line 330) doesn't specify which exact file to look at. - Line 127. "We have used 500 µmol · s−1m−2 as the default light intensity of AL." For simulations, an intensity of 500 µmol m−2s−1 was used, but for experiments (line 152) "The intensity of red AL was set at approx. 457 µmol m−2s−1". We understand that matching the actinic light during the experiment to 500 µmol m−2s−1 cannot be possible, alternatively we suggest that the simulations be carried out at 457 µmol m−2s−1 for sake of consistency. Importantly, is 457 µmol m−2 s−1 the value given by the manufacturer for the chosen setting, and did you measure it to confirm its value? (depending on instrument calibration, usage and age, the light output can be different than set) - Line 204, 205. "The calculated steady-state NPQ values are higher for SP intensities below 3000 µmol·s−1 m−2", according to Fig.5, it seems that the threshold is rather 2000, than 3000 (or 4000). - Fig7. To test the "actinic effect" of SP duration, we would suggest to perform a simulation with AL=100 µmol m−2 s−1 AL and/or AL=0 to check whether SP themselves can induce NPQ.According to Fig8A (experimental), it seems that at 0.8s, NPQ is indeed slightly higher than with shorter SP duration. - Line 370, a necessary addition would be to list here, or write a template of, what you suggest for minimum information is needed as standard for the community. It could be similar to Table 2, and needs to include duration of AL on, off and AL quality. Minor comments - Line 46. "Allow", should be "allows" - Line 75. "Groups but also" should be "groups experimentally, but also" - Line 115. Replace higher by vascular. - Line 140. 26C is higher than standard temperature for Arabidopsis growth (22C), what's the rationale for choosing this temperature? - Line 150. Define Fv and explain if the 5s of far red light is turned on at the very beginning of the experiment i.e. before time 0. - Line 153. "default settings (10)", specify "set at value of" 10. We suggest writing a small table with these parameters (see major comment). - Line 161. Which leaf did you choose, younger or older? This information is important to state, see differences with leaf age for example in Bielczynski et al. Plant Phys (2017) doi: 10.1104/pp.17.00904. - Line 173-174. We suggest that the SP time points are moved to the methods section. - Line 185-186. "In the upper panel….derived NPQ and ՓPSII", this whole sentence can be removed as it should be clear from the figure legend. - Line 211. "Far more" how many did you look at? - Fig. 6. "6A and 6A". Should be "6A and 6B" - Line 234. "Switching on AL with the first SP in light-triggered after 1 s" suggest rewording as it was unclear what light-triggered means. - Line 241. The observed effect is likely due to the total conversion of zeaxanthin to violaxanthin for long periods of dark-adaptation. - Line 243. Suggest changing "whereas" should be "however" as it is clearer. - Line 256. Define PMST. - Line 264-268. We suggest moving this block of text to the discussion section. - Line 264. "AL is another important information" should be "AL is another important piece of information" - Fig. 8B and 8D. As the simulated curves seem to all overlap, and often in this study we look for fine nuances between data, we think it would be beneficial to read the simulated curve superimposed on top of the experimental data allowing a fair comparison and analysis between the two types of data. Displaying the same graphs at a larger scale would help to read them. To help in this, we propose Figure 8 to be divided in two figures, since Fig. 8A-D is related to "SP experiment" while Fig. 8E-H is related to the "delay experiment". This would allow the size of the panels to be increased to help the reader interpret the data. - Fig. 8F and 8H. Plot titles "Delay NPQ/ՓPSII Sim lation", should be "Delay NPQ/ՓPSII Simulation" - Fig. 9 seems to be redundant as the reader should be able to observe the difference between the two independent experiments by comparing Figure 8A and 8E. We therefore suggest that Fig. 9 be removed. - Line 286. "Measurements are" should be "measurements have been" - Line 289-303. We suggest moving this block of text to the introduction section - Line 324. Replace "many" by "all"! - Line 351. "Agreements" should be "agreement" - Line 361-372. We feel that the points made in this block of text have already been made earlier in the manuscript and repeated several times. Therefore this block of text can probably be omitted as it is redundant. - General comments concerning the figures: we suggest adding dark/light bars to the top of most plots in Figures 3B-C, 4B-C, 6A-D, 7A-B, 8A-H; as it would improve the readability/interpretation of the plotted data. Fig. 2-8, figure identifier letters are presented in a different font style than the rest of the text, throughout the document. While we recognize them to be hyperlinks, we think font style should be uniform. Jack Forsman and Andre Graca (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Jingfang Hao, Maria Paola Puggioni, Pierrick Bru, Aurélie Crepin, Wolfgang Schröder.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5884374. Schumacher et al. discuss the metabolic pathways evolved by green plants to degrade chlorophyll molecules. The authors combined a large-scale comparative phylogenomic approach with biochemical characterization of putative novel phyllobilins to shed light on how the degradation pathway evolved. This manuscript points to the evolution of chlorophyll degradation, in particular the later detoxification steps, having accompanied the green lineage’s transition to land. An extensive list of orthologous genes from a diverse number of species was identified. The manuscript stands out for the wide evolutionary view on the chlorophyll degradation pathway, which is neither an easy nor a common research subject. The in silico approach for phyllobilins identification is quite innovative and will surely give great hints for biomolecules discovery. A comprehensive bioinformatics work has been done to carefully identify and select the genes analyzed in the manuscript and the related phylogenetics figures are outstanding. Although this work is of great interest, we have some comments that could be addressed in the next version. Major comments We would suggest to tone down the title as it may be that less or no chlorophyll catabolites were detected in mosses, charophytes and chlorophytes due to the smaller number of species analyzed (Fig.5), and some of these clades may have evolved other phyllobilins exporting and modifying proteins. Please discuss these possibilities. Page 9, line 16: regarding the identification of 15 putatively novel phyllobilins, mass spectrometry data together with in silico produced list of diagnostic ions are presented to support these structures. Would it be possible to provide additional confirmation via a standard (an internal or a synthesized one) or alternatively to state which other molecules these m/z plus profiles could be corresponding to? How likely is it that they correspond to other unrelated compounds? Page 5, line 11, comment on two species lacking CAO. Are they lacking chlorophyll b? Minor comments Intro, page 3, line 19, explain why it is unlikely that nitrogen remobilization be a conserved evolutionary trigger. Page 8, line 15, what is the carbon source used for heterotrophic growth? Line 19, instead of “etc” list all conditions tested. Page 9, line 24, could you discuss or introduce whether oxidations are spontaneous or enzymatic? Could you comment in the discussion about the significance of Chl breakdown catabolites in cellular signaling from an evolutionary point of view? Methods, in the paragraph “Plant material growth and chlorophyll degradation induction”, page 15, lines 1-12: several of the growth conditions are missing e.g. at which temperature S. moellendorfii was growing? At which temperature and humidity was the dark incubation of leaves performed? In the Sup. Figure 6 B, the structure of phyllobilin 3b is not shown. It could be nice to have that too or to explain why it is not shown. Please, also include the relevant spectra from the remaining identified compounds as supplemental data (assuming that the 4 presented spectra/structures are also of novel compounds). Maybe we missed it, but where can we find the identifiers of the genes that were used in the gene trees shown Figures 2, 3 and 4? The Sup. Table 1 is first cited in the Methods. Maybe, cite it in the introduction (page 4, line 12) in order to cite it chronologically with respect to Sup. Table 2. Page 9, lines 3 and 23, “SFig. 6” should read SFig. 4. Page 5, line 34, please indicate the gene names corresponding to the abbreviation for SGR2 and SGL. Minor grammatical errors and typos are present in the text, e.g. page 24: line 24, “loss” should be “lost” line 29, “one orthogroups” should be “one orthogroup” line 33, “do belongs” should be “does belong” line 35, “expect” should be “except” line 36, “have derived a pre-existing enzyme” should be “have derived from…” Domenica Farci, Sam Cook (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Maria Paola Puggioni, Aurélie Crépin, André Graça, Jack Forsman, Jingfang Hao, Pierrick Bru, Jianli Duan.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5884376. The manuscript by Ruiz-Sola et al. investigates the relationship between photoprotection responses, carbon concentrating mechanisms (CCM) and CO2 availability in Chlamydomonas reinhardtii. While photoprotection responses, mediated by LHCSR3, LHCSR1 and PSBS, are traditionally described as triggered by excess of light, this manuscript highlights the role of intracellular CO2 levels (both deriving from the environment and from mitochondria metabolism) in regulating these responses. Indeed, it demonstrated that photoprotection, and especially LHCSR3-mediated responses, are from one side inhibited in conditions in which inorganic carbon is largely available and abundant (acetate and external CO2 supply) and on the other side induced in conditions of reduced CO2 availability. Furthermore, CCM are also induced under high light (HL), in response to a drop in intracellular CO2 levels due to increased photosynthetic carbon fixation. While changes in the expression levels of both LHCSR3 and CCM genes at different CO2 concentration and under HL respectively, were previously reported, this manuscript has the novelty to connect these observations in an elegant experimental set up with several genetic backgrounds to confirm and prove their hypothesis through the use of mutants affected in mitochondrial respiration and of metabolic modeling. The proposed model for light-independent regulation of photoprotection is convincing and solidly backed-up by data. In addition a role for CIA5 in positively regulating LHCSR3 (and to a lesser extent PSBS) mRNA expression and in negatively regulating LHCSR1 at the post-transcriptional level is shown. However, we have some comments and suggestions to improve the manuscript, listed below. Major comments Figure 3, and corresponding result paragraph pages 6 to 8: - A large part of the results (1.5 pages) focuses on modelling the interaction between acetate metabolism and intracellular CO2 levels. Although we are not experts in mathematical modeling and thus we are unable to give proper feedback regarding this part of the paper, we think it adds small value to the main results of the paper. This is especially true as the modelling relies on a number of assumptions (listed at the bottom of page 7) which are not supported by literature nor experimental data, weakening the solidity of its conclusions. As it is, only assumption iv (page 7, "the acetate uptake is low (...) for the mutants (as indicated in Fig 2C and F)" is backed up by data. We suggest moving figure 3 to Supplementary material and shorten its description in the results and discussion. Please also provide better support to justify the assumptions i to iii, as well as the assumption that photon uptake is not altered in the mutants (e.g. do they have similar chlorophyll content?) and make the conclusions more solid. - Page 6, "In line with the experimentally observed values, we found that the predicted generation times for the icl and dum11 strains (...) did not differ from those of LL grown WT cells". Please, provide the experimental values for the mutant strains, or rephrase the sentence. In Figure S1F to K: - During exposure to L2, the basal fluorescence Fo’ in the presence of acetate (and to a lesser extent CO2) is rising together with the maximal fluorescence Fm’. Please provide explanation or hypotheses for this fact, and if it might or not affect ETR and NPQ calculations. Also consider replacing "qE" with "fast-induced fluorescence quenching" or simply "NPQ", as other regulation mechanisms might affect these fluorescence measurements. - Please precise the time points you used for assessment of Fo, Fm, and calculation of qE. To make this figure more understandable please provide clearer fluorescence traces in Figure S1 (C-K), showing only Fo, Fm and Fm' (ideally one plot for each genotype to be consistent with Y(II) and NPQ plots, L-N and O-Q) and a separate panel with Fo and Fo'. Figure 6B and corresponding text page 11: - Please provide an explanation for the cia5 mutant line accumulating high LHCSR1 protein and not fully reverting to wild type level in the complementation line under VLCO2 (and dark/ air). This aspect needs to be taken into account and clarified, especially in light of CIA5 proposed role as LHCSR1 regulator at the post transcriptional level. Rephrase this sentence "However, LHCSR1 protein over-accumulated in the cia5 mutant under all conditions tested, although the WT phenotype was only partially restored in cia5-C (Fig. 6B)" as this the case only for HL/air. Minor comments Title: Please add "algal" to the title, or a similar clarification. Introduction - Page 3, when mentioning carbonic anhydrases (CAH) as part of the CCM please list the ones involved in CCM. Not all CAH are part of CCM (also it is useful to see their names, since the expression levels of some of them are measured in the results part). - Page 4, in the sentence "Here, using genetic, transcriptomic and mathematical modelling approaches, we demonstrate that the inhibition of LHCSR3 accumulation and CCM activity by acetate is at the level of transcription and a consequence of metabolically produced CO2" please replace "transcriptomic" with "expression analysis on selected genes", since no transcriptomics work has been shown in this manuscript. - Page 4, please reformulate the sentence "This work emphasizes the critical importance of intracellular CO2 levels in regulating LHCSR3 expression and how light mediated responses may be indirect and reflect changes in internal CO2 levels resulting from light intensity dependent, photosynthetic fixation of intracellular CO2". Based on the previous reports and from this work, we can say that internal CO2 levels are important in regulating activation and inhibition of LHCSR3-photoprotection mechanisms, BUT it does not mean that the light effect is indirect, this has not been proved yet. Furthermore, photoprotection by NPQ could lead to diminished CO2 fixation rate (especially sustained "photoinhibitory" quenching types), thereby increasing internal CO2 concentration which would according to your model repress photoprotective genes. This could be the case for genes involved in qE but may not be a general rule for "photoprotection". The title could also reflect that aspect by specifying NPQ, qE in lieu of photoprotection. qRT-PCR results: - qRT-PCR results are described here as "mRNA accumulation". Please replace this nomenclature with "relative expression levels" or "relative gene expression". - It is stated in the methods, page 17, that the results presented are normalized on a reference standard gene, GBLP. However, the results presented seem to be (also?) normalized on the WT LL air. Is this correct? If so, please precise or clarify it. Instead of normalizing the data to the WT LL air, we suggest normalizing the transcript abundance of the target genes in each sample to your internal reference standard gene (GBLP) only. - Please provide a description on how the relative gene expression levels were calculated. We suggest calculating by determining the ΔCt levels of the sample compared to the standard and the 2^(-∆Ct) as final value. Paragraph "LHCSR3 transcript accumulation is impacted by acetate metabolism": - page 4, it is not clear in here the transition between TAP and HSM media. - page 4, rest of the text and figures legends, please indicate CO2 concentration in ppm (according also to figure 6D) instead of 5% CO2. - icl-C line not behaving the same. Paragraph "CO2 generated from acetate metabolism inhibits accumulation of LHCSR3 transcript and protein": - Page 5, "RHP1 (...) encodes a CO2 channel shown to be CO2 responsive and to accumulate in cells growing in a high CO2 atmosphere". It is unclear here if RHP1 is sensitive to intracellular, extracellular, or both levels of CO2. Please better describe how the protein levels reflect the intracellular CO2 concentration. - Since Figure 1 includes results both described in this and in the previous paragraph, we suggest grouping the results described in Fig1 in a single paragraph and make a shorter but clearer description of the results. - Fig 1: you could merge Fig 1A and C in a single plot with WT icl, icl-C and dum 11 in LL and HL to make the comparison between the mutants clearer. Also, the same can be done for the panels B and D. Paragraph "Impact of carbon availability in other qE effectors" - Page 8, "We took HL acclimated cells that typically accumulate both LHCSR3 and LHCSR1 proteins (Fig. S2A) and performed photosynthetic measurements in the absence or presence of 20 mM sodium bicarbonate; the bicarbonate addition was just before performing the photosynthetic measurements. As expected, bicarbonate enhanced rETR (Fig. S2B) and….almost completely suppressed qE despite the fact both LHCSR3 and LHCSR1 had accumulated in the cells (Fig. S2)". The accumulation of these proteins was not checked in presence of bicarbonate in this particular experiment (the bicarbonate was added shortly before measuring photosynthetic parameters). Please, rephrase the sentence. - Page 9 and Figure 4B and Figure 5C " PSBS protein accumulation could not be evaluated because it was not detectable under the experimental conditions used. " It is surprising you could not detect PSBS in these conditions (600 uE), while it was possible in the conditions described in Fig 6B. At least the HL conditions (600 uE) were the same in these two experiments. Please provide an explanation for this, or if it is not possible, rephrase without mentioning PSBS expression and accumulation in the text and for clarity reasons remove Fig4A. Paragraph "CCM1/CIA5 links HL and low CO2 responses" - Page 9, "To elucidate the molecular connection between photoprotection and CCM, we analyzed mRNA accumulation from the CCM genes encoding LCIB and LCIE (involved in CO2 uptake), HLA3, LCI1, CCP1,CCP2, LCIA, BST1 (Ci transporters), CAH1, CAH3, CAH4 (carbonic anhydrases) and the nuclear regulator LCR1, all previously shown to be strongly expressed under low CO2 conditions (see (49)for a review on the roles of each of these proteins and (45)for the more recently discovered BST1)." Please provide the whole name for the reported abbreviation of the proteins that were not mentioned earlier in the text. Paragraph "Intracellular CO2 levels regulate photoprotective and CCM gene expression in the absence of light" - Page 11 and Figure 6C: the figure is unclear, making the quantification hard to pick up and understand. Please consider replacing the "LHCSR3 (r.u.)" line above the panel by a histogram clearly displaying the LHCSR3/ATPB ratio; add error bars. If no repeats/error are available, please refrain from using these quantification data and rephrase the paragraph page 11 to replace quantitative statements ("...which was reflected by a 3-fold change in the accumulation of the protein…", "and 21 fold (protein) compared to air dark conditions (Fig. 6A-C)...", "...and protein level (by a factor of~9)...") by qualitative ones. - Page 11, "This CIA5-independent regulation of mRNA in the presence of light could account for the contribution of light signaling in LHCSR3 gene expression, possibly via phototropin (10)" This should be discussed properly in the discussion section. - Page 11, "the cia5 mutant did not accumulate significant amounts of LHCSR3 protein under any of the conditions tested (Fig. 6B)" The lack of LHCSR3 in HL in the cia5 mutant is quite striking considering that its transcript level is quite high and similar to wild type. Please provide a possible explanation for this observation. - Page 12, please replace " in accord" with "in line" or "it fits the hypothesis" - Page 12, Fig 6E, for clarity, please develop the statement "In contrast to LHCSR3, sparging with VLCO2 only partly relieved the suppression of transcript accumulation for the CCM genes in the presence of DCMU (Fig. 6E)". For instance, consider adding "..., bringing it back to LL levels instead of the accumulation observed in HL in the control (see dotted line in Fig. 6E)". Discussion - Page 13, "Increased CO2 levels were found to dramatically repress LHCSR3 mRNA accumulation, in agreement with previously published works (34, 35), but had little impact on accumulation of LHCSR1and PSBS transcripts". It is hard to say if it has a little or no impact on PSBS gene expression. We suggest not putting emphasis on the PSBS expression levels difference. - Page 14, beginning of last paragraph, "Our data demonstrate that most of the light impact on LHCSR3 expression is indirect". Please tone down these sentences and discuss them with regards to the recent study by Redekop et al. (ref. 46). We suggest replacing this sentence with "Our data demonstrate that besides LHCSR3 gene expression variation together with changes in the light environment, it is also tightly linked to CO2 intracellular changes". - Page 14 "It is tempting to propose that CO2 could be considered as a retrograde signal for remote control of nuclear gene expression, integrating both mitochondrial and chloroplastic metabolic activities". This sentence is very speculative, although clearly marked as such. To further soften the point, please consider adding "Further studies will have to be carried on to confirm or infirm this possibility". - Page 15 "The CIA5-independent light-dependent induction of photoprotective genes possibly involves phototropin, as previous shown (10), but may also involve retrograde signals such as reactive species (46, 77). Our findings also highlight the need to develop an integrated approach that examines the role of CO2 and light, with respect to CO2 fixation, photoreceptors, and redox conditions on the regulation of photoprotection and to consider photoprotection in a broader context that includes various processes involved in managing the use and consequences of absorbing excess excitation". If you want to discuss photoprotection relationships with photoperception etc you should give more context, otherwise it is not easy to catch for people who are not familiar with this possible connection. The data of this manuscript do not show any experiments related to photoperception, yet and it has been mentioned in four times in the paper. In our opinion this does not fit in the discussion of this manuscript. - Data S2A, please replace "reaction names" by "enzyme names". - Figures S1C to K, Figure S2C, Figure S4A to C, it is stated that the fluorescence is normalized to Fm, when it seems to be normalized to the maximum fluorescence reached during the experiment (highest Fm’ point). Please correct either the figures or the legend. - Figure S2B, it is stated that the statistical analyses are shown in the graph, though they appear to be missing. Maria Paola Puggioni and Aurélie Crepin (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Jingfang Hao, André Graça, Pierrick Bru,Jack Forsman.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5778995. The manuscript by Seydoux et al. investigates the role of proton potassium antiporter KEA3 in diatoms. The authors first demonstrated the pH dependence on photoprotection, specifically non photochemical quenching (NPQ) and showed that NPQ can be induced in the dark by acidic pH. They found that KEA3 modulates NPQ by impacting the proton motive force (PMF); indeed generated kea3 mutants showed increased partitioning into deltapH. Importantly they showed that diatom KEA3 in contrast to plant KEA3 possesses an EF hand motif which can bind Ca2+ and proposed that it controls KEA3 activity. The role of KEA3 and pH in affecting the NPQ response has been previously shown in other photosynthetic organisms however the novelty of this study lies in the demonstration that NPQ can be induced in the dark by acidic pH and the proposed role of Ca2+ in regulating KEA3 function. Major comments - Page 5, you state that pH-induced quenching in the dark was accompanied by the conversion of DD into DT. Please provide de-epoxidation state (DES) at t15 time (Fig. 1B) to substantiate this statement. Starting DES would also be informative to ensure there was no retention of DT/zeaxanthin in the dark. - Also to ensure there is no sustained NPQ (and/or damage or disconnected antenna) at t0, please provide Fo and Fm levels for all NPQ kinetics experiments. Assessing PSII accumulation by D1 immunoblot could be done to ensure PSII damage does not occur. - In Fig. 2F, it is not clear which data points represent HL or ML treatment as well as which ones come from light or dark period. Please indicate them in different colors or symbols. Also clarify whether you have averaged data from the kea3 mutant alleles. - To confirm that lack of complementation by deltaEF is not due to mislocalization, please show whether deltaEF accumulates at the thylakoid membrane. Minor comments - Page 3, Introduction, specify qE after NPQ response; PSBS should be written PsbS - Page 4, DD-dependent NPQ should be DT-dependent - Page 4, we suggest changing "crucial" to "Given the unknown role" if pH-dependence of NPQ in diatoms hasn't been fully established before - Page 8, KEA3 most likely homolog, were there other homologs than the two shown in Fig. S5? also discuss conservation of other ion channels (is Phatr J11843 thylakoid-localised?) and if they could compensate for the absence of KEA3 in KO mutant (by being upregulated for instance). - Fig2B, comment on the band at ~80kDa in OE, is that from cleavage of GFP? - Fig2G, shouldn't you expect a lower dpH in the OE? Please comment. - Page 13, for the statement that only dpH can modulate NPQ, we would suggest to tone down or specify that this is the assumption made here as it could be that dpsi modulates NPQ but has yet to be shown! - Most of the protein analyses were performed loading samples based on protein content, when possible please provide proof that chlorophyll levels are comparable between the genotypes (at least for the native gels) - Abstract, extra 'of' between capacity and via; page 23, extra 'being' between likely and less important - Define acronyms when used for the first time - There is a lot of 'peculiar' in the text - Fig. 2D, star symbol instead of square symbol, check consistency of symbols Pushan Bag, Pierrick Bru (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Maria Paola Puggioni, Jingfang Hao, Jack Forsman, Wolfgang Schröder, Emma Cocco, Jianli Duan.