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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/6415043. The manuscript "The role of LHCBM1 in non-photochemical quenching in Chlamydomonas reinhardtii" by Liu et al. aims to elucidate how LHCBM1 is involved in non-photochemical quenching (NPQ) in Chlamydomonas reinhardtii. The Chlamydomonas mutant lacking LHCBM1 (npq5) displays a low NPQ phenotype. The authors found that the antenna size and LHCSR3 accumulation are not responsible for the lower NPQ phenotype in npq5. They also artificially acidified the lumenal pH to protonate LHCSR3 for NPQ induction and found that npq5 NPQ is still low. They propose that absence of LHCBM1 could alter the association of LHCSR3 with the PSII supercomplex or that LHCBM1 interacts with LHCSR3 which would enhance its quenching capacity. This work enriches the knowledge about the impact of lack of LHCBM1 on antenna size, PSII function, LHCSR1 and 3 proteins accumulation and NPQ capacity during a 48-h high light treatment. Major comments - Fig. 1, it is stated that LHCBM1 (Type IV) does not accumulate but Fig. S1 and S2 show that Type IV accumulates between 7 and 9% of the total amount of LHCBMs. Could you comment on this accumulation, and whether it increases in high light? - Statistical test between WT and npq5 for all the data represented in bar graphs would be required to state that differences are significant. Consider showing all data points in addition to error bars. - Consider providing Fo and Fm values for all fluorescence measurements (and D1 accumulation) to discuss whether a possible increased Fo in npq5 in HL is due to disconnected antenna and/or loss of functional core complexes. - Discussion, discuss why there is much less LHCSR1 in the npq5 mutant? Is this regulation at the transcriptional or post-translational level? It could be that LHCBM1 interacts with LHCSR1 preventing its degradation. In the abstract, and discussion, as no interaction data is shown, consider toning down statements regarding LHCBM1 interaction with LHCSR3. - Discussion, discuss whether similar results were observed for the npq4 mutant (increased antenna size in HL and less functional PSII observed in npq5). Is this phenotype linked to the low NPQ capacity or specific to the lower level of LHCBM1 in the npq5 mutant? Minor comments - Please give a title to summarize the first result in Fig. 1. In the result section, consider making the section subtitles more informative about the main result from that section (e.g., "Photoprotection capacity after high-light acclimation" could be "NPQ induction is severely hampered in npq5 after high-light acclimation"). - Page 3, method, actinic light of 1,500 μmol photons.m-2.s-1 (and saturating pulses of 12,000 μmol photons.m-2.s-1) are used for the fluorescence measurements. Comment on the choice of such a high light intensity (and high intensity pulses, actinic effect?). Why not use a light intensity closer to the HL treatment? Also state length of dark-acclimation prior to each type of measurement. - Immunoblot method, state dilutions used and secondary antibody type and dilution used. - Page 4, it is stated that "the level of the other LHCBMs is similar to that of WT (CC-425)" by referring to Fig. S1 and S2. It is difficult to see in Fig. S1 that the level of other LHCBMs is similar in npq5 and WT as the data is represented in percentage of total LHCBMs. Could you also represent the amount of the different LHCBMs in npq5 normalized to WT in a bar graph? For the Type III and Type II/I LHCBMs accumulation, a dilution series might be best for quantification to ensure that CP47 antibody signal is not saturated. In Fig. S1, npq5 should be italic. In Fig. S2, one of the labels is likely wrong: there are two 48h time points. By biological replica, is it meant three independent batches of grown and treated cells? What is the fourth band right below the upper band detected by the LHCBM5 antibody at 48h? - Fig. 5, please enlarge panels B and C to match panel A. - Fig. 6, specify in the legend that 24h and 48h refer to the HL treatment. - Discussion, "and confirmed here by analysis of the [npq5] mutant" (not npq4) Jingfang Hao and 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, Aurélie Crepin, Fadime Demirel, 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/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/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.

During our search for potential templates of HIV-1 reverse transcriptase (RT) and integrase (IN) dual inhibitors, the methanolic extract obtained from aerial parts of Limonium morisianum was investigated. Repeated bioassay-guided chromatographic purifications led to the isolation of the following secondary metabolites: myricetin, myricetin 3-O-rutinoside, myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside, (-)-epigallocatechin 3-O-gallate, tryptamine, ferulic and phloretic acids. The isolated compounds were tested on both HIV-1 RT-associated RNase H and IN activities. Interestingly, (-)-epigallocatechin-3-O-gallate and myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside potently inhibited both enzyme activities with IC50 values ranging from 0.21 to 10.9 μM. Differently, tryptamine and ferulic acid exhibited a significant inhibition only on the IN strand transfer reaction, showing a selectivity for this viral enzyme. Taken together these results strongly support the potential of this plant as a valuable anti HIV-1 drugs source worthy of further investigations.