CYP20-3 deglutathionylates 2-CysPRX A and suppresses peroxide detoxification during heat stress

Posttranslational modification by a reduced glutathione determines distinctive quaternary structures and functions between two 2-cysteine peroxiredoxin A and B in the chloroplasts.

required to appreciate the "real world" relevance. The occasionally somewhat confusing organization of the manuscript (it is simply difficult to read) together with the description of the experiments contributes to this impression.
That being said, if the authors can make their manuscript more accessible and also provide in vivo support for their mechanistic in vitro work, this manuscript would make an outstanding contribution on how 2CPs and GSH contribute to signalling in plants!! Specific comments: 1. Abstract and introduction are very specific and technical and require the reader to be familiar with 2CPs and their regulation in order to understand the background to the work. It should not be hard for the authors to make the introductory section more accessible to the reader which would also significantly raise the impact of their work.
2. Another somewhat confusing aspect of the introduction is that Figure S1, S2 and S3 are referenced in the introduction -i.e. as background information. I got the impression that this was novel data and should thus be described in the results section and the introduction. If this is validation of previously published data this should be highlighted better. In the results section I found it hard to immediately understand for all experiments whether they represent an in vivo approach to elucidate the regulation of 2CPs or an in vitro reconstitution of regulatory mechanisms. It can be figured out, however, it would be preferable if this would be immediately understandable for the reader. 3. "This mutant 2CPBV106I/I109V indeed behaved like 2CPA" (page 6 of the manuscript): This is an exciting finding. How about the corresponding point mutations in 2CPA? Could that convert it into a "2CPB-like behaviour"? Along the same lines: can this be replicated in vivo by transforming the point mutation constructs into 2cpa or 2cpb mutant plants? This should be doable since the authors have mutant lines for 2cpa and 2cpb as well as an antibody recognizing both proteins ( Figure S2A and B). Would the point mutation constructs in the difference 2cp mutant plant lines complement their phenotypes (for example hydrogen peroxide production in 2cpa)? 4. Figure 1, panels C and D: I assume "GST" should be GSH? 5. "The latter further supports a unique and autonomous activity of GSH in activating redox signaling, independently from GSSG-mediated Sglutathionylation and its oxidative signaling (Xiong et al, 2011;Grek et al, 2013), that relays metabolic signaling during the stress-responsive activation of defense and acclimation pathways." (page 7 of the manuscript) -this is only based on the (in vitro) 2CP data and should either be rephrased to reflect that or supported by in vivo evidence. 6. "Herein, TRX and NTRC exhibited minimal reductase activity, exhibiting little if any effect on the quaternary structure of 2CPsGS." (page 8 of the manuscript) - Figure 3 panel D suggests that addition of NTRC leads to more 2CPB icosamer. Is that correct? If yes this could rephrased, if not perhaps a clearer image with better resolution could be supplied. 7. "Though, it was noticeable that the supplement of SRX causes a slight delay in the gel mobility of partial 2CPBsGS (Fig 3F), indicating that SRX may be able to target the S-S bond forming 2CPBGS icosamers ( Fig 1D)." (page 8 of the manuscript): Would 2CPB cysteine mutants (C -> S) be helpful to visualize this better? In panel 3F I can only observe the decamers but not really the icosamers. Generally, the icosamer is nicely visible in Figure 1 but very hard to see in the other figures. 8. "Indeed, the deglutathionylation of 2CPAGS paralleled an attenuation of its (peroxidase activity, reducing H2O2 ( Fig 5A)" (page 10 of the manuscript): The experiment has been carried out under "heat stress" but no control under ambient temperatures is shown. 9. "The enhanced redox capacity in turn coordinates the expression of a subset of ODPAresponsive genes (ORGs), including HSP17.6, as well as CYP81D11 (Fig 5D and Appendix Fig S9)." (page 10 of the manuscript): The results in Figure 5D suggest that HSP17.6 is only partially dependent on OPDA (or the action of CYP20-3). I suggest to use more than two marker genes for the action of OPDA.
10. "These results concur with the conclusion that CYP20-3-dependent OPDA (defense) signaling counteracts the photosynthetic (growth) mechanisms, drawing a new insight into the mode of "growth and defense trade-offs" that optimizes energy productions and distributions during defense mechanisms (e.g., HS responses), maximizing plant growth and survival processes." (page 10 of the manuscript): I don't precisely follow the authors argumentation here. There is little data on the connection to OPDA signalling in the manuscript at present. This needs to be much better explained to the reader and possibly supported by experimental evidence. 11. Technical note: qRT-PCR experiments are normalized to a single reference gene. An experimental design using at least three reference genes has been recommended for past years and for a high-quality publication should be standard procedure.

Referee #2 Review
Report for Author: First of all, I would like to state that I was not aware at the time of reading the manuscript that this had already been rejected and my reviewing it was within the procedure of rebuttal/re-evaluation. Thus, I was not influenced by either the first editorial decision or the rebuttal by the authors.
Liu et al describe a mechanism by which a chloroplastic cyclophilin is involved in the growth/defense response of plants under heat stress. The authors state that OPDA as a stress-response molecule binds to CYP20-3 and thereby leads to decrease of 2CPA peroxidase activity and at the same time the increase of SAT1 action to build up thiol synthesis. General remark regarding the quality of the manuscript: The text is often hard to read and the use of words often in the wrong context, e.g. "metabolize" is not a process that changes protein structures but usually refers to catabolizing something such as glucose to ATP in glycolysis. Another example is "entertaining" plants at 12h light/12h dark -I very much doubt that growing in climatic chambers is entertaining to the plants. There are more cases like this, which confuse the reader.

Introduction:
The introduction is well linked to the rest of the manuscript, but I miss several facts that should be mentioned here. First the connection of 2CP and NTRC (especially since there are results later regarding their interaction), second the confirmed and important role of OPDA in hormone signaling. Researchers from that field will wonder upon reading the manuscript why it is not mentioned anywhere that OPDA is the precursor of jasmonic acid and thus heavily involved in stress response to cold, wounding and biotic stresses. It needs to be discussed how OPDA is sequestered to the different biochemical processes -binding to CYP20-3 would surely prevent export and conversion to jasmonate. Results: Figure 3/RNA expression: the authors argue their case with co-expression of RNA and deduce protein activity from that. This is simple not feasible. It has been shown many times now (among others by the Dietz lab, from whom they even got material) that RNA amount has in ~50% nothing to do with protein amount, let alone protein activity. Co-expression data can be valuable nonetheless, but this needs to be discussed and the phrasing adapted. The same holds true for Figure 5.
Discussion: please refer to my comments above! Regarding the novelty of the presented data, details about 2CP function in relation to OPDAbinding to CYP20-3 are indeed new as is the differentiation of 2CPA and 2CPB. This manuscript points to a yet unrecognized role of these components in the growth/defense regulation of plant met abolism. However, there are point s arguing against publicat ion in this journal -the met hods used here are quit e basic, repet it ive and not in any way sophist icat ed. The result s will only be int erest ing to a specialized reader communit y from the plant field. Thank you for transferring your manuscript entitled "CYP20-3 Deglutathionylates 2-CysPRX A and Suppresses Peroxide Detoxification during Heat Stress" to Life Science Alliance. The manuscript was assessed by expert reviewers at another journal, and the editors transferred those reports to us with your permission.
The reviewers who evaluated your study elsewhere appreciated your findings, but would have expected a further reaching demonstration of their (potential) in vivo relevance. This concern does not need to get fully addressed for publication in Life Science Alliance, and we would thus like to invite you to submit a revised version of your manuscript to us, based on the reviewer reports already at hand. Please provide a point-by-point response and accordingly drastic changes to the manuscript text (both reviewers). The requests for controls and technical comments should get addressed (rev#1), too.
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Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Authors' Response to Reviewers July 6, 2020 We are very pleased to submit our revised manuscript (#LSA-2020-00775-T, Liu et al.) for consideration of publication in your esteemed journal, Life Science Alliance. We wish to sincerely thank the referees for their very constructive comments and concerns. All these have been addressed in the revised manuscript. In addition, the text of the manuscript has been significantly revised for clarity and proficiency pursuant to your editing. Please find our responses to the referees below.

Response to Referee #1
We have addressed the referee's comments and concerns regarding the sufficient introduction and discussion. Below we have described all the changes and modifications made in the revised manuscript as per the suggestions. A = our answer/response to the reviewer's suggestions/comments which are lettered in lowercase (e.g. a, b, c, etc).
a. Abstract and introduction are very specific and technical and require the reader to be familiar with 2CPs and their regulation in order to understand the background to the work. It should not be hard for the authors to make the introductory section more accessible to the reader which would also significantly raise the impact of their work. A, We agree with the referee's suggestion, and have revised and substantially rewritten both abstract and introduction to be more accessible for readers. These changes/insertions include; 1) "The latter is a thiol-based peroxidase, protecting and optimizing photosynthesis by reducing its toxic byproducts (e.g., H2O2)", (page #2 line #8 in the revised manuscript).
2) "The peroxidase cycle starts by their redox-active cysteine (Cys; the peroxidatic Cys, CP) in the catalytic center is oxidized to sulfenic acid (CP-SOH) by a broad set of peroxide substrates. The CP-SOH residue then reacts either with another Cys of the same or an adjacent subunit, or with another thiolcontaining compound, to form an inter-or intramolecular disulfide (C P -S-S-C R) , which is later reduced 3) "However, 2CPs are known to typically form an obligatory homodimer as the CP from one monomer is connected via a disulfide bond to the resolving Cys (CR) located at another monomer. The oxidation of Cys P then, in consequence, deactivates the catalytic activity of 2CPs. Thus, 2CP dimers require electron donors such as an NADPH-dependent thioredoxin reductase C (NTRC), thioredoxins (TRXs) and/ or cyclophilin 20-3 (CYP20-3), which reduces (activates) them to be able to metabolize the detoxification of a toxic byproduct in photosynthesis (i.e., H2O2), and the activation of Calvin cycle enzymes such as a fructose 1,6-bisphosphatase ( b. Another somewhat consuming aspect of the introduction is that Figure S1, S2 and S3 are referenced in the introduction -i.e. as background information. I got the impression that this was novel data and should thus be described in the results section and the introduction. If this is validation of previously published data this should be highlighted better. In the result section I found it hard to immediately understand for all experiments whether they represent an in vivo approach to elucidate the regulation of 2CPs or an in vitro reconstitution of regulatory mechanisms. It can be figured out, however, it would be preferable if this would be immediately understandable for the reader. In agreement, our preparatory analyses exhibited that 2CPs can uniquely bind a negatively charged tripeptide GSH, a major nonprotein thiol-antioxidant in plants (Fig S1A). GSH-binding (hereafter, called GSH-glutathionylation) then differentially modulates the conformational states of 2CPs (Figs 1A lane 1,  2, 5 and 6, and S2), stimulating the monomerization and peroxidase activity of 2CPA, while decamerizing and accentuating the chaperone activity of 2CPB (Fig S3, Lee et al, 2015)", (page #5 line #22 in the revised manuscript).
c. "This mutant 2CPBV106I/I109V indeed behaved like 2CPA" (page 6 of manuscript): This is an exciting finding. How about the corresponding point mutations in 2CPA? Could that covert it into a "2CPB-like behaviours"? Along the same lines: can this be replicated in vivo by transforming the point mutation constructs into 2cpa and 2cpb as well as an antibody recognizing both proteins ( Figure S2A and B). Would the point mutation constructs in the difference 2cp mutant plant lines complement their phenotypes (for example hydrogen peroxide production in 2cpa)? A, As per the referee's suggestion, we have examined and presented the behavior of a WT and mutant (I106V/V109I) 2CPAs upon GSH-glutathionylation (Fig 1B lanes 13 and 14 in the revised manuscript), exhibiting 2CPB-like pattern.
We also agree that the complementation study is a logical next step to further validate the physiological role of 2CPA. However, because of current circumstantial and time constraints, we have discussed with an editor, and instead substantiated the role of 2CPA by using the second mutant allele (2cpaII; MPMI 25:294) as well as a double KO mutant (2cpa/2cpb; J Ext Bot 61:4043). These results clearly underpinned the important role of 2CPA in the removal of H2O2 during heat stress responses ( Fig 5B in the revised manuscript). We hope this is acceptable to the reviewer.
d. Figure 1, panels C and D: I assume "GST" should be GSH?. A, The type-error has been corrected in the revised Figure 1.
e. "The latter further supports a unique and autonomous activity of GSH in activating redox signaling, independently from GSSG-mediated S-glutathionylation and its oxidative signaling (Xiong et al. 2011; Grek et al, 2013), that relays metabolic signaling during the stress-responsive activation of defense and acclimation pathways." (page 7 of the manuscript) -this is only based on the (in vitro) 2CP data and should either be rephrased to reflect that or supported by in vivo evidence. A. As per the referee's suggestion, we have presented new ex vivo experiment, demonstrating that CYP20-3-dependent OPDA signaling and subsequent increases in GSH:GSSG ratio (PNAS 11:9559) play a critical role in regulating the conformational state (rates) of 2CP GS to accentuate its peroxidase activity during wound defense and acclimation processes ( Fig S5 in the revised manuscript). We have also rephrased the last sentence, as it reads in the revised manuscript (page #8, line #8) "The latter further supports a unique and autonomous activity of GSH in activating redox signaling, independently from GSSG-mediated S-glutathionylation and its oxidative signaling (Xiong et al, 2011; Grek et al, 2013), that relays hormone (e.g., OPDA; Fig S5) signaling during the stress-responsive activation of defense and acclimation pathways." f. "Herein, TRX and NTRC exhibited minimal reductase activity, exhibiting little if any effect on the quaternary structure of 2CPs GS ." (page 8 of the manuscript) - Figure 3 panel D suggests that addition of NTRC leads to more 2CPB icosamer. Is that correct? If yes this could rephrased, if not perhaps a clearer image with better resolution could be supplied. A. We appreciate referee's suggestion. We have repeated the assay, and also reassessed earlier results.
To the end, we have carefully concluded that NTRC showed little effect on the icosameric structure of 2CPB GS , and thus -as per the referee's suggestion -replaced the image with one of newer data with an improved resolution ( Fig 3D in the revised manuscript).
g. "Though, it was noticeable that the supplement of SRX causes a slight delay in the gel mobility of partial 2CPBs GS (Fig 3F), indicating that SRX may be able to target the S-S bond forming 2CPB GS icosamers ( Fig 1D)." (page 8 of the manuscript): Would 2CP cysteine mutants (CàS) be helpful to visualize this better? In panel 3F, I can only observe the decamers but not really the icosamers. Generally, the icosamer is nicely visible in Figure 1 but very hard to see in the other figures. A, We appreciate referee's insights. We -first of all -have replaced the Fig 3F with one with an improved resolution.
On the other hand, we re-examined and found that a mutant 2CPB does not improve the visuality of 2CPB GS icosamers. As a referee suggested, we have validated that 2CPB cysteine mutant (converting C 175 to S) is able to form icosamers upon the GSH-glutathionylation ( Fig  1A in this letter). However, the level icosamerization of 2CPB•C175S GS was similar to that of WT 2CPB GS ( Fig  1B in this letter). Moreover, we have detected that the initial amounts of icosameric WT and mutant 2CPBs GS (these are shown in Fig 1 in the revised manuscript) were noticeably reduced by the dialysis process for the following experiments (Fig 1B in this letter; and these are shown in Figs 3 and 4 in the revised manuscript). Taken together, we conclude that 2CPB•C175S GS does not significantly improve the visuality of 2CPB GS icosamers, and believe that further studies are needed to comprehend the biochemical characteristics and stability of 2CPB GS icosamers.
h. "Indeed, the deglutathionylation of 2CPA GS paralleled an attenuation of tis peroxidase activity, reducing H2O2 ( Fig 5A)" (page 10 of the manuscript): The experiment has been carried out under "heat stress" but no control under ambient temperatures is shown. i. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes (ORGs), including HSP17.6, as well as CYP81D11 (Fig 5D and Appendix Fig S9)." (page 10 of the manuscript): The results in Figure 5D suggest that HSP17.6 Is only partially dependent on OPDA (or the action of CYP20-3). I suggest to use more than two marker genes for the action of OPDA. A, As per the referee's suggestion, we have examined and presented the attenuation of another OPDA marker gene (HSP70, Plant Cell 20:768) in cyp20-3 KO mutant during heat stress (Fig S10B in the  revised manuscript).
j. "The results concur with the conclusion that CYP20-3-dependent OPDA (defense) signaling counteracts the photosynthetic (growth) mechanisms, drawing a new insight into the mode of "growth and defense trade-offs" that optimizes energy productions and distributions during defense mechanisms (e.g., HS responses), maximizing plant growth and survival processes." (page 10 of the manuscript): I don't precisely follow the authors argumentation here. There is little data on the connection to OPDA signaling in the manuscript at present. This needs to be much better explained to the reader and possibly supported by experimental evidence. A. We agree with the referee's comment, and removed the previous discussion on the role of OPDA signaling. The revised manuscript now reads (page #10 line #24) "These results support the versatile activity of CYP20-3 in OPDA signaling, which conveys the activation of disease resistance against Alternaria brassicicola, and defense responses to different abiotic stresses such as wounding and HS (Park et al, 2013; Figs 5D and Fig S10)". k. Technical note: qRT-PCR experiments are normalized to a single reference gene An experimental design using at least three reference genes has been recommended for past years and for a high-quality publication should be standard procedure. A. As per the referee's suggestion, we have examined and presented the qRT-PCR experiments (revised Figs 3G, 3H, 5C, 5D, and S10; revised Materials and Methods, page #17 line #11), normalized against three reference genes, polyubiqutin (UBC), glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A (PP2A), and evaluated using qbasePLUS 3.0 (Ramakers et al, 2003).

Response to referee #2
a. The text is often hard to read and the use of words often in the wrong context, e.g. "metabolize" is not a process that changes protein structures but usually refers to catabolizing something such as glucose to ATP in glycolysis. Another example is "entertaining" plants at 12h light/12 dark -I very much doubt that growing in climatic chambers I entertaining to the plants. There are more cases like this, which confuse the reader. A, As per referee's suggestion, we have replaced "metabolize" with "convert" (page #8, line #14 in the revised manuscript), and "entertained" with "grown" (page #9 line #1 in the revised manuscript), and carefully revised the manuscript.

Introduction:
b. The introduction is well linked to the rest of the manuscript, but I miss several facts that should be mentioned here. First the connection of 2CP and NTRC (especially since there are results later regarding their interaction), second the confirmed and important role of OPDA in hormone signaling. Researchers from that field will wonder upon reading the manuscript why it is not mentioned anywhere that OPDA is the precursor of jasmonic acid and thus heavily involved in stress response to cold, wounding and biotic stresses. It needs to be discussed how OPDA is sequestered to the different biochemical processes -binding to CYP20-3 would surely prevent export and conversion to jasmonate. A. As per the referee's suggestions, we have introduced (1) the connection of 2CP and NTRC and (2) the role of OPDA in hormone signaling in the revised introduction. In addition, we have discussed (3) the impact of OPDA binding to CYP20-3 in the revised discussion. 1) The revised manuscript reads (page #3, line #7) "Thus, 2CP dimers require electron donors such as an NADPH-dependent thioredoxin reductase C (NTRC), thioredoxins (TRXs) and/or cyclophilin 20-3 (CYP20-3), which reduces (activates) them to be able to metabolize the detoxification of a toxic byproduct in photosynthesis (i.e., H2O2), and the activation of Calvin cycle enzymes such as a fructose 1

3)
In page #13, line #5: "Note that our jasmonate quantifications in cyp20-3 KO mutants (Park et al, 2013) suggest that, in a resting states, CYP20-3 could sequester OPDA and reduce downstream jasmonate productions, but the increased accumulations of OPDA under stress conditions could circumvent the effect of its binding to CYP20-3, exhibiting little difference in JA accumulations between WT and cyp20-3, together proposing that OPDA and JA signaling are activated in parallel and/or accumulatively in defense responses."

Results:
c. Figure 3/RNA expression: the authors argue their case with co-expression of RNA and deduce protein activity from that. This is simple not feasible. It has been shown many times now (among others by the Dietz lab, from whom they even got material) that RNA amount has in ~50% nothing to do with protein amount, let alone protein activity. Co-expression data can be valuable nonetheless, but this needs to be discussed and the phrasing adapted. The same holds true for Figure 5. A. We agree with the referee's suggestion and have incorporated additional descriptions. The revised manuscript reads (page #9, line #4) "Caveat is that 2CPs are highly abundant (~0.6% of the total plastid proteins), and determined to exhibit slow-turnover rates (Horling et al, 2003;Dietz et al, 2006). Hence, the co-expression of 2CPs and SRX may not, at once, tie in their physiological and functional interactions." In addition, (page #10, line #16) "This explains the HS-induced accumulation of, already abundant, 2CPB GS (Fig 5C) that constitutes a stable, decameric conformation (Fig 1) conferring chaperon activity ( Fig S3C)." Having addressed all of the concerns of the reviewers, we hope that the revised manuscript is now acceptable for publication in Life Science Alliance. Thank you for submitting your revised manuscript entitled "CYP20-3 Deglutathionylates 2-CysPRX A and Suppresses Peroxide Detoxification during Heat Stress". Your manuscript was re-reviewed by one of the original referees at another journal, and their report is attached below. We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The authors have addressed most of my comments to my satisfaction or provided convincing reasoning why a particular experiment would be beyond the scope of the manuscript. The manuscript is also considerably easier to read compared to the previous version.
My only remaining suggestion, which has not been addressed is, that I would prefer the text in the results section and the figure legends to mention clearly when an experiment has been carried out with bacterially produced protein. As in the previous version of the manuscript, the reader can figure this out, however, it takes time. This will be very very easy to address without negatively impacting the manuscript or the presentation of the work.
Few grammatical errors should also be corrected, for example Materials and Methods -> Preparation of recombinant proteins -> To prepare tag-free version, purified 2CPs and mutant 2CPBs were incubated with thrombin protease, and remove the His-tag -> this likely should be "to remove the His-tag". Other example: Section title "CYP20-3 block the peroxidase activity..." should be "CZP20-3 blocks the peroxidase activity...".