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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/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. |
