Primary Characterization of a Life-Cycle Mutant akasusabi of the Red Alga Neopyropia yezoensis
Round 1
Reviewer 1 Report
The manuscript by Mikami, Matsumura and Yamamoto address an interesting topic on seaweed biology that is to deepen in the life cycle of the red seaweed Pyropia yezoensis which is also one of the main species farmed for nori. The approach is very appropriate since isolating life cycles from mutants is a proper way for bringing to light the mechanisms that control the generation transition in rhodophytes. One of the main achievements is that a life cycle of a red seaweed mutant is reported. Therefore, in my opinion, the research is well based and the achieved results are sound.
I will list first some general comments and later on more specific ones.
- The introduction is adequate and updated, reflecting that the authors know the topic well.
- M&M are properly detailed and explained. The results section talks about growth rate (line 141). How do growth rate was measured? It is not stated in M&M section. It seems to me that only temporal changes in the length of the thallus are used as a proxy for growth? If it is correct, then would be better to talk of growth instead of growth rate, since (specific) growth rate use to be approached from an exponential model and units would be better (time-1).
- Results discussion are correct
- Discussion: I fully agree that a karyotipic analysis of chromosome numbers in mutant aks should be done.
- Page 292. I do not know that discoloration al 0ºC is due to photoinhibition, since light in culture is quite below light saturation point for this species
Some specific comments:
- Check throughout the text that aks is in italics.
- The first time a species in cited in a paragraph the name should be written complete (Pyropia yezoensis instead of yezoensis).
- line 93: better photon flux density than irradiation
- line 94: provide the temperature in parenthesis
- line 101: provide the brand of the spectrophotometer
- line 103: phycocyanin (lowercase)
- line 105: different font (ºC)
- line 141: As commented above, you did not estimate growth rate (as specific growth rate as it should be usual). You measured the thalli length over time.
- line 142: were? (Instead of was)
- line 144: higher (Instead of high)
- line 150-152: a bit confuse sentence. Please rewrite it.
- line 195: and in WT (after aks)
- line 255: carpospores
- line 299: a bit confuse sentence. Please rewrite it.
- line 334-335: capital letters?
Author Response
We sincerely applicate Reviewer 1 providing valuable comments useful for manuscript revision. According to these comments, we did our best for revision. Additional or revised sentences and words are indicated by red characters in the revised version of the manuscript.
Responses to Reviewer 1
The results section talks about growth rate (line 141). How do growth rate was measured? It is not stated in M&M section. It seems to me that only temporal changes in the length of the thallus are used as a proxy for growth? If it is correct, then would be better to talk of growth instead of growth rate, since (specific) growth rate use to be approached from an exponential model and units would be better (time-1).
Response: Supplementary Figure 1 was revised to show the growth rate. We also added a sentence “Growth rate of WT and mutant was calculated by dividing the length of germilings (cm) of asexually released monospores by duration of incubation (day) under 0, 4, 10, 15, 20 and 25°C for 4, 11, 15 21, 28 and 36 days.” at lines 96-98 in the revised version. Accordingly a subtitle of 2.2 was changed to “Algal Strain, Culture Conditions and Calculation of the Growth Rate” in the revised manuscript.
Discussion: I fully agree that a karyotipic analysis of chromosome numbers in mutant aks should be done.
Response: We also recognize the significance of karyotipic and DNA content analyses. Since cells of Bangiales are too small to apply for ploidy analysis using the regular system of flow cytometry, karyotype analysis is the best way to analyze ploidy of the aks sporophytes. In fact, we have already tried to perform this type of experiments; however, we have to improve our technique to obtain results. For example, staining of chromosomes with the fluorescent CYBER Gold had a problem by the presence of many symbiotic bacteria. We think that the karyotype analysis is one of our next subjects for characterization of the aks mutant.
Page 292. I do not know that discoloration al 0ºC is due to photoinhibition, since light in culture is quite below light saturation point for this species
Response: we revised “photoinhibition” to “inhibition of nitrogen uptake resulting in destruction of phycobilisomes” at lines 286-287 in the revised manuscript. Accordingly, references [36-38] was changed to [36] by citing Takahashi, M., Kumari, P., Li, C. and Mikami, K. Low temperature causes discoloration by repressing growth and nitrogen transporter gene expression in the edible red alga Pyropia yezoensis. Mar. Environ. Res. 2020, 159, 105004 with changed in reference numbers of original 39-51 to 37-49 in both text and Reference part.
Some specific comments:
Check throughout the text that aks is in italics.
Response: We checked aks as Italic in the manuscript.
The first time a species in cited in a paragraph the name should be written complete (Pyropia yezoensis instead of yezoensis).
Response: We believe that species name should be written completely as Pyropia yezoensis in the first time of the text. From next time, P. yezoensis is OK except for figure legends.
line 93: better photon flux density than irradiation
Response: It was changed as suggested.
line 94: provide the temperature in parenthesis
Response: We added “(0, 4, 10, 15, 20 and 25°C)”.
line 101: provide the brand of the spectrophotometer
Response: We added “ (GENESYS 10 Bio, Thermo Electron Corporation, Beverly, MA, USA)” at lines 104-105 in the revised version.
line 103: phycocyanin (lowercase)
Response: It was changed as suggested at line 107 in the revised version.
line 105: different font (ºC)
Response: Correct font was used at line 109 in the revised version.
line 141: As commented above, you did not estimate growth rate (as specific growth rate as it should be usual). You measured the thalli length over time.
Response: Please see above.
line 142: were? (Instead of was)
Response: It was changed as suggested at line 146 in the revised version. Accordingly, “rate” was changed to “rates” at line 145 in the revised version.
line 144: higher (Instead of high)
Response: It was changed as suggested at line 148 in the revised version.
line 150-152: a bit confuse sentence. Please rewrite it.
Response: We think the word “macroscopic” is responsible for confusion. Thus, this word was removed at line 155 in the revised version.
line 195: and in WT (after aks)
Response: Since the sentence is compare findings between aks and WT, adding “and in WT” is not necessary.
line 255: carpospores
Response: It was changed as suggested at line 259 in the revised version.
line 299: a bit confuse sentence. Please rewrite it.
Response: We revised a sentence as “Wounding and heat stress promote asexual reproduction in aks, but the frequency of promotion was influenced differently by these stresses” at lines 294-295 in the revised manuscript.
line 334-335: capital letters?
Response: Names of gene products usually written by capital letters, especially for those of genetically identified genes at least in plants.
Reviewer 2 Report
The manuscript describes a new strain of Pyropia yezoensis with disrupted mitosis in the gametophyte-to-sporophyte transformation that was obtained via selection of spontaneous mutant in long-term
culture. The text provides good background and description of the significance of such contribution to the pool of algal cell-cycle mutants, and describes the morphoogical differences in the various
stages of aks mutant vs wildtype in great detail while providing a wealth of illustrative photographic figures. To my surprise and dissapointment, however, no genetic data is presented, despite the fact that at least some of the genes potentially involved in cell cycle regulation (i.e. genes which may contain the mutation/s in their sequence or vicinity) are known from P. yezoensis (as mentioned in the discussion) or other algae and basic experiments which could screen at least some of these loci (such as PCR + product length comparison and/or Sanger sequencing, possibly RACE-PCR to check the UTRs) would be relatively easy to perform. Authors also propose, that the sporophytes produced by the mutant line could be diploid and point towards potential future karyotyping that is necessary to confirm this statement that is quite central to the understanding of the significance of the aks strain's difference from the wildtype. Again, in this case I would expect some attempt to estimate the ploidy of the organism (e.g. rough estimation of DNA content/cell or, if available and affordable, using telomeric probes). In summary, the manuscript is solid and sound in its method description and result presentation, but it lacks, in my opinion, in data from other than simple observational methods - and acquisition and presentation of such data could make it much more impactful and well-founded in its interpretations and discussion.
Regarding my suggestions to the text itself:
- I suggest mentioning the origin of the mutant (i.e. that it is natural-occuring and not artificially generated) earlier in the text (currently on line 82) so that the reader doesn't get a wrong idea
- line 123: which stress does the sentence refer to? It seems to relate to the controls mentioned in the previous sentence - and I'm unsure of the meaning.
- line 128: "excepting" -> "except"
- line 138: missing space
- line 141: aks should be italicized
- line 144: "high" -> "higher"
- line 146: I'm not familiar with the term "shrink-and-twist" morphology - is this something that is commonly used in phycological writing? I wasn't able to determine to what exact morphological changes does this description refer to based on the text and the figure legend (which doesn't mention this term)
- line 157: "both 3" -> either "both" or "all 3"?
- line 160: does the horizontal cell divison mean something else than anticlinal? If yes, please specify; if not, unify the terminology
- line 164: The convergence of both cell types to a similar size is interesting. Could this point towards a general maximum cell size constraints of this lifestage/organism/group? What else is known about this topic, and could it maybe be worth mentioning in the discussion?
- line 179-180: Does this mean only the aks mutant was able to do this under such conditions? Was this unexpected?
- lines 190-191: I'm slightly confused by the logical structure of the sentence, perhaps a language unhandiness? Please rephrase so that it is clearer what is notable/surprising about this.
- Figure 3 legend: I would prefer more detailed legend that would be more descriptive of the differences that should be noticed in the photographs as they may be less obvious to readers that are less familiar with the studied organism.
- Figure 5: I believe "Top" in the headline of D panel should read "Tip"? Also, please add some division between the partitions of the x axis as in the current state it looks linge a single axis with nonsensical numbering.
- Figure 5 legend: I suspect the A, B, and C mentioned on lines 214-215 are obsolete legends for "top", "middle", and "bottom" barplots under D, as they clearly do not refer to the actual panels A, B, and C?
- line 224: what does the "this" refer to? the temperature? please clarify the sentence.
- line 229: "calluses were produced between 10 and 20°C, in contrast to WT where they were produced only at 25°C" - does this mean that they were *only* produced at 10-20°C in aks or at 25°C, same as the WT, and *additionaly* at the lower temperatures too? please clarify.
- lines 235-237: the first sentence is a mouthful - maybe rephrase to make it more digestable? For example, it is gramatically unclear whether the "producing" in "produce carpospored producing" refer to the verb or the object?
- line 245: double negative, "no enlarged spores did not appear" => either "no enlarged spores did appear" or "enlarged spores did not appear"
- lines 257-258: maybe suggest how this could be achieved? is this a realistic proposition for near future study or more of an abstract idea for the future?
- line 267-268: How was the haploidy of the thalli determined? Also, please explain the reasoning behind why does this means that the sporophyte can be diploid?
- lines 288-289: Any ideas on why only phycoerythrin and not the other pigments? Was this difference in pigment production under increased photosynthetic activity described elsewhere?
Author Response
We sincerely applicate Reviewer 2 providing valuable comments useful for manuscript revision. According to these comments, we did our best for revision. Additional or revised sentences and words are indicated by red characters in the revised version of the manuscript.
Responses to Reviewer 2
To my surprise and dissapointment, however, no genetic data is presented, despite the fact that at least some of the genes potentially involved in cell cycle regulation (i.e. genes which may contain the mutation/s in their sequence or vicinity) are known from P. yezoensis (as mentioned in the discussion) or other algae and basic experiments which could screen at least some of these loci (such as PCR + product length comparison and/or Sanger sequencing, possibly RACE-PCR to check the UTRs) would be relatively easy to perform.
Response: Yes, we recognize significance of gene expression analysis of putative life cycle-regulating genes found previously. However, information about nucleotide sequences of these genes was very short (small unigenes) and thus incomplete (meaning not full-length), which prevented design of primes for gene expression analysis. Thus, we now try to obtain full-length cDNAs for genes of interest. We think that experiments to obtain genetic data are the next step of our study.
Authors also propose, that the sporophytes produced by the mutant line could be diploid and point towards potential future karyotyping that is necessary to confirm this statement that is quite central to the understanding of the significance of the aks strain's difference from the wildtype. Again, in this case I would expect some attempt to estimate the ploidy of the organism (e.g. rough estimation of DNA content/cell or, if available and affordable, using telomeric probes).
Response: We also recognize the significance of karyotipic and DNA content analyses. Since cells of Bangiales are too small to apply for ploidy analysis using the regular system of flow cytometry, karyotype analysis is the best way to analyze ploidy of the aks sporophytes. In fact, we have already tried to perform this type of experiments; however, we have to improve our technique to obtain results. For example, staining of chromosomes with the fluorescent CYBER Gold had a problem by the presence of many symbiotic bacteria. We think that the karyotype analysis is one of our next subjects for characterization of the aks mutant.
In summary, the manuscript is solid and sound in its method description and result presentation, but it lacks, in my opinion, in data from other than simple observational methods - and acquisition and presentation of such data could make it much more impactful and well-founded in its interpretations and discussion.
Response: Your above points are very important and we have already recognized these importance. We are now trying to resolving these problems in the aks study. We however felt difficulty to progress experiments without enough man power. My laboratory was in fact moved to the new affiliation in the last year and most of lab members disappeared.
We have revised our manuscript according to your valuable suggestions to improve it as mentioned in below.
I suggest mentioning the origin of the mutant (i.e. that it is natural-occuring and not artificially generated) earlier in the text (currently on line 82) so that the reader doesn't get a wrong idea
Response: We added “, but not artificially generated,” at line 82.
- line 123: which stress does the sentence refer to? It seems to relate to the controls mentioned in the previous sentence - and I'm unsure of the meaning.
Response: To avoid confusion, words “stress treated” were removed at line 126 in the revised version.
- line 128: "excepting" -> "except"
Response: It was changed as suggested at line 132 in the revised version.
- line 138: missing space
Response: It was changed as suggested at line 142 in the revised version.
- line 141: aks should be italicized
Response: It was changed as suggested at line 145 in the revised version.
- line 144: "high" -> "higher"
Response: It was changed as suggested at line 148 in the revised version.
- line 146: I'm not familiar with the term "shrink-and-twist" morphology - is this something that is commonly used in? I wasn't able to determine to what exact morphological changes does this description refer to based on the text and the figure legend (which doesn't mention this term)
Response: Yes, "shrink-and-twist" is not commonly used, because there is no manuscript describe this phenomenon in Bangiales as far as we know. The meaning of this word is very simple; leafy gametophyte represents abnormal shape with shrinking and twisting. Panel A of Figure 2 shows this phenotype especially in aks under 25℃. Supplementary Figure 3 represents formation of abnormal multi-cell layer is responsible for "shrink-and-twist".
- line 157: "both 3" -> either "both" or "all 3"?
Response: The word “both” was changed to “all” at line 162 in the revised version.
- line 160: does the horizontal cell divison mean something else than anticlinal? If yes, please specify; if not, unify the terminology
Response: The word “anticlinal” was employed at line 165 in the revised version.
- line 164: The convergence of both cell types to a similar size is interesting. Could this point towards a general maximum cell size constraints of this lifestage/organism/group? What else is known about this topic, and could it maybe be worth mentioning in the discussion?
Response: As far as we know, there is no report indicating changes in the size of carpospores after releasing to date. Alternatively, we unexpectedly recognized enlargement of cell size of carpospores in WT through analyzing those of aks mutant. Although we cannot answer if the cell size is maximum in the species and Bangiales, we proposed that the lack of cell dividing activity in carposporangium in aks suggests inability of production of carpospores in this mutant. We discuss about this point in the second paragraph of the Discussion.
- line 179-180: Does this mean only the aks mutant was able to do this under such conditions? Was this unexpected?
Response: Yes, production of conchocelis from blades is now found in only aks among Pyropia species at present and thus unexpected.
- lines 190-191: I'm slightly confused by the logical structure of the sentence, perhaps a language unhandiness? Please rephrase so that it is clearer what is notable/surprising about this.
Response: Honestly, we cannot get a point of this comment. We believe there is no language unhandiness in this sentence.
- Figure 3 legend: I would prefer more detailed legend that would be more descriptive of the differences that should be noticed in the photographs as they may be less obvious to readers that are less familiar with the studied organism.
Response: In my experience, detailed explanation in figure legends usually commented to remove these sentences, because the text explains the details. We hope to escape from such a comment from, for example, the Reviewer 1 if we revised the legends. Indeed, the text represents the details of results.
- Figure 5: I believe "Top" in the headline of D panel should read "Tip"? Also, please add some division between the partitions of the x axis as in the current state it looks linge a single axis with nonsensical numbering.
Response: We revised Figure 5 by correcting the word and problem in the X axis.
- Figure 5 legend: I suspect the A, B, and C mentioned on lines 214-215 are obsolete legends for "top", "middle", and "bottom" barplots under D, as they clearly do not refer to the actual panels A, B, and C?
Response: We removed A, B and C from the legend to panel D as lines 215-216 in the revised manuscript.
- line 224: what does the "this" refer to? the temperature? please clarify the sentence.
Response: The word “this” was changed to “which” at line 228 in the revised manuscript.
- line 229: "calluses were produced between 10 and 20°C, in contrast to WT where they were produced only at 25°C" - does this mean that they were *only* produced at 10-20°C in aks or at 25°C, same as the WT, and *additionaly* at the lower temperatures too? please clarify.
Response: Expression was changed to “at 10 and 20°C in addition to 25°C” at line 233 in the revised version.
- lines 235-237: the first sentence is a mouthful - maybe rephrase to make it more digestable? For example, it is gramatically unclear whether the "producing" in "produce carpospored producing" refer to the verb or the object?
Response: We revised “to produce carpospores producing sporophtes from vegitative cells of gametophytes” to “to generate carpospores from vegetative cells of gametophytes for production of sporophytes” at lines 240-241 in the revised manuscript.
- line 245: double negative, "no enlarged spores did not appear" => either "no enlarged spores did appear" or "enlarged spores did not appear"
Response: It was changed to “no enlarged spores appeared” at line 249 in the revised manuscript.
- lines 257-258: maybe suggest how this could be achieved? is this a realistic proposition for near future study or more of an abstract idea for the future?
Response: We deleted this sentence to avoid making a confusion (please see line 261).
- line 267-268: How was the haploidy of the thalli determined? Also, please explain the reasoning behind why does this means that the sporophyte can be diploid?
Response: Although ploidy of thalli and sporophyte is our speculation, haploidy of the thalli is generally accepted. Thus, question seems to be the reason why we speculate diploidy in sporophyte. We did not obtain direct evidence of diploidy; however, as indicated at lines 268-272 in the original manuscript, it has been suggested that Banhgiales has an ability to duplicate its genome spontaneously during generation transition from gametophyte to sporophyte. Thus, we speculate a possibility of diploidy in sporophyte produced by apogamy. Such a sporphyte indeed produces carpospores to develop gametophyte in a normal way. However, to obtain a direct evidence, we have to observe ploidy by karyotype analysis or DNA content analysis. Please see our second response to your comments in above.
- lines 288-289: Any ideas on why only phycoerythrin and not the other pigments? Was this difference in pigment production under increased photosynthetic activity described elsewhere?
Response: We deleted the sentence.
Reviewer 3 Report
The manuscript entitled "Primary characterization of a life-cycle mutant akasusabi of the red alga Pyropia yezoensis" addresses a relevant topic about reproduction in the "akasusabi" mutant.
The theme is suitable for this journal and the interest for readers is high.
I believe that it should be published after the introduction of the taxonomic corrections indicated below.
Corrections needed:
line 3 (Title) - red alga Neopyropia yezoensis
line 16/17 - Neopyropia yezoensis
line 18 - Conchocelis (in italics)
line 20 - Conchocelis (in italics)
line 23 - Conchocelis (in italics)
line 28 - N. yezoensis
line 29 - Neopyropia yezoensis
line 34 - Conchocelis (in italics)
line 53 - Neopyropia yezoensis (formerly Pyropia yezoensis)
line 72 - Neopyropia yezoensis
line 83 - N. yezoensis
line 90 - N. yezoensis
line 168 - Neopyropia yezoensis
line 174 - Neopyropia yezoensis
line 183 - Conchocelis (in italics)
line 198 - Neopyropia yezoensis
line 206 - Neopyropia yezoensis
line 239 - N. yezoensis
line 264 - N. yezoensis
line 273 - Neoporphyra haitanensis
line 297 - N. yezoensis
line 321 - N. yezoensis
line 331 - N. yezoensis
line 335 - Physcomitrium patens (formerly Physcomitrella patens)
line 338 - N. yezoensis
line 345 - N. yezoensis
Author Response
We sincerely applicate the Reviewer 3 providing comments useful for further revision of our revised manuscript. We revised the previous version of our manuscript according to these comments. Words changed are indicated by red characters in the current version of the manuscript.
Responses to Reviewer 3
The manuscript entitled "Primary characterization of a life-cycle mutant akasusabi of the red alga Pyropia yezoensis" addresses a relevant topic about reproduction in the "akasusabi" mutant.
The theme is suitable for this journal and the interest for readers is high.
I believe that it should be published after the introduction of the taxonomic corrections indicated below.
Corrections needed:
line 3 (Title) - red alga Neopyropia yezoensis
line 16/17 - Neopyropia yezoensis
line 18 - Conchocelis (in italics)
line 20 - Conchocelis (in italics)
line 23 - Conchocelis (in italics)
line 28 - N. yezoensis
line 29 - Neopyropia yezoensis
line 34 - Conchocelis (in italics)
line 53 - Neopyropia yezoensis (formerly Pyropia yezoensis)
line 72 - Neopyropia yezoensis
line 83 - N. yezoensis
line 90 - N. yezoensis
line 168 - Neopyropia yezoensis
line 174 - Neopyropia yezoensis
line 183 - Conchocelis (in italics)
line 198 - Neopyropia yezoensis
line 206 - Neopyropia yezoensis
line 239 - N. yezoensis
line 264 - N. yezoensis
line 273 - Neoporphyra haitanensis
line 297 - N. yezoensis
line 321 - N. yezoensis
line 331 - N. yezoensis
line 335 - Physcomitrium patens (formerly Physcomitrella patens)
line 338 - N. yezoensis
line 345 - N. yezoensis
Response:
Although we do not know whether a current phylogenic classification of previous Pyropia species is widely accepted, the species name was corrected as Neopyropia yezoensis in the manuscript as mentioned. The word “Conchocelis” was changed in italics.
In addition, we changed “the aks mutant” at lines 26-27 in the previous version to “AKS” at line 27 in the current version. In addition, “the aks gene” at line 348 in the previous version was also changed to “AKS” at line 348 in the current version.
We also correct other simple typing errors in the text. Since these corrections are deletion of characters or brackets, these are not indicated by red characters.
Round 2
Reviewer 2 Report
I appreciate the changes in text and authors' explanation for the lack of genetic and karyotyping data which is completely understandable, and I'm excited to hear that they will hopefully be soon published in a follow-up article, as some of the experiments are already underway. I therefore find the manuscript satisfactory in its current form.
Author Response
Response:
We are very happy hearing from you about accepting our current situation and would like to continue analysis of the aks mutant as you suggested.
