Pseudostichococcus Stands Out from Its Siblings Due to High Salinity and Desiccation Tolerance

Round 1

Reviewer 1 Report

This paper addresses two abiotic stressors: desiccation and high salinity, which are related in terms of their effects on cellular water homeostasis. 
The authors examined the synergistic effects of salinity on desiccation on various representative taxa within the Stichococcus clade. Results showed that unlike other Stichococcus taxa, Pseudostichococcus was able to fully recover from desiccation with and without salinity stress. This observation was associated with increased proline production under salinity stress and a higher proline:sorbitol ratio in Pseudostichococcus compared to siblings.

The topic undertaken is interesting, the issue raised in the paper is worthy of investigation.
The authors have correctly written the introduction, correctly stated the purpose of the paper. The literature was selected appropriately, the authors used the latest reports.

However, the following things should be improved in the work:
1. Materials and Methods - How was the effective quantum yield of PSII (Y(II), ΔF/Fm') of each strain measured? 
2. In the description of statistical analyses, it states that Pearson's cointegration analysis was performed. There is too little data of this analysis in the description of results and discussion.
3. In my opinion, the discussion presented in the paper is just an extended description of the results. Please present information from the literature and compare your results with that. 
4. There are no conclusions in the paper.
Minor comments are marked in yellow in the manuscript.

Comments for author File: Comments.pdf

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

Although this work does not strike me as a breakthrough study, it is a nice piece of algal stress physiology which suits nicely the scope of the journal. the research is carefully desinged and carried out. The manuscript is generally well-written, so it can be accepted; the minor style flaws can be easily removed during copyediting.

Author Response

Dear Reviewer,

 

Thank you very much for your kind review of the manuscript. Minor stylistic changes and corrections, as well as suggestions from the other reviewers, have been made.

 

Best regards,

 

Ms. Anh Van

Dr. Karin Glaser

Reviewer 3 Report

Van & Glaser compare the proline content, the proline/sorbitol ratio and dissection/rehydration (yield based) response of nine different strains from the Stichococcus clade. The overarching research question – how does a high salinity impact the desiccation response of these ‘terrestrial’ algae?- is interesting and relevant. The manuscript is overall well-written and I particularly enjoyed the build-up of the introduction. My main concerns are (1) some essential information (e.g. measurement of sorbitol, standardisation of proline, calculation of recovery values) is missing and some numbers in the papers (e.g. nr of strains tested, light intensity during desiccation experiment) probably need to be corrected, (2) several of the conclusions are not supported by the results and a (3) substantial overlap with previously published work of the same authors (https://doi.org/10.3390/microorganisms9091816). I hope all these issues can be remediated without too much work.

 

General remarks:

There are important limitations to what conclusions can be drown. Since no growth was measured of the strains, the amount of osmolytes cannot directly be related to survival at higher salinities and remains speculative. Since only a few strains were used, with all strains other than Pseudostichococcus being only represented by one strain, no conclusions can be drown about species specific responses and the discussion should focus on strain specific effects. Furthermore, since more strains of the Pseudostichococcus genus have been included, it is far more likely that at least a few of these will respond differently. You can of course still speculate on how the observed strain effects might reflect species attributes, but please be careful.

Growth data are however present in https://doi.org/10.3390/microorganisms9091816 where also a nearly identical desiccation/recovery experiment is performed. Sorbitol and sucrose were measured instead of proline. The main novelty of this study is therefore in the proline concentrations, which is also the main focus of the discussion. I think the paper would benefit from a more explicit comparison/merger between both studies: are desiccation/recovery values identical, do the growth rates observed in the 2021 paper support the statements made in the discussion (at first glans, the growth of Pseudostichococcus strains is not so much better at higher salinities). If the reported measurements of this study have directly been recovered from the 2021 study, this needs to be clearly stated and discussed accordingly.

A broad phylogenetic introduction of the Stichococcus clade would be nice in the introduction

Should the Absolute Salinity be expressed as g / kg?

The sources/brands of chemicals are missing, whilst this is less relevant for common salts, this is good to have for f.i. the proline standard

The proline content is determined for each culture, but the biomass was not standardised? Without standardising the absolute proline concentration per culture to the number of cells (or another biomass proxy, like F0) it is very difficult to interpret proline values.

The method on how sorbitol concentrations have been determined is not given

It is unclear how the recovery values from the rehydration experiment are calculated and compared. No repeated measures anova is used, so I assume only one recovery value per replicate is obtained? No statistics on the proline measurements are reported

Although the layout of the graphs is very nice, it is not always straightforward to come to the same conclusions as the authors. I think figure 1 & 2 would benefit a lot from also including the species names (or intials). Having the strains in the same order in both figures would also be nice. Having the 0Sa and the 30Sa ratios joined per strain instead of in the different graphs would make it easier to see the trends per strain. If a statistical comparison between strains is done, please also indicate significant differences in the graphs

Figures 3 and 4 are very nice, but it is nearly impossible to compare 0Sa to 30Sa. I think it would be a lot easier to compare these effect if the data are grouped per stain (a plane with 2 lines).  Please indicate a gap on the x axis of the recovery graphs

 

Minor remarks:

L13: to -> two

L13: studied the effect of salinity

L17: proportion -> ratio

L17/18: to the other strains tested

L47: double spacing

L48: focused heavily on species

L50 : this statement requires a more recent publication

L53-54: remove ‘and the relative…agriculture’

L60-61: reference to support this statement?

L61: L.Moewus, 1951

L62: Stichococcus Nägeli 1849

L63: double spacing

L82: fifteen -> nine!

L85 µm ->µmol

L90: what do you mean with ‘authentic’

L93-95: please clarify how the recipe was amended to create the ‘90 SA 3N BBM+V medium’ (amount & type of salt

L122: remove ‘respectively’

L13: 40µmol is not low-light, it is even higher than the cultivation conditions, typo? ‘high’ light in combination with desiccation can be harmful to the cells and is certain to impact the PAM measurements

L127: how long after the dehydration experiment was the rehydration experiment initiated

L130: I assume this is the 0Sa medium for all treatments?

L150: from strains within Pseudostichococcus -> from Pseudostichococcus strains

L152: please quantify the ‘slight’ increase

L155: Pseudostichococcus in italics

L193: In general, data salt-induced -> Data of salt..

L195: data -> experiments

L196: well-known -> known

L196-197: you do not have the data to support this statement, e.g. sucrose and ribitol were not measured

L197: Stichococcus  strains under high salinity

L201: potentially allows

L209-210: no differential halotolerance proven here

L211: Pseudostichococcus and Tritostichococcus strains but

L211-212: not proven that these are more salt-sensitive, they just should a less proline production

L230-232: I don’t really know what you mean with this statement. Is halotolerance not always an adaptation in this context.

L240-241: strains proportionally increased proline

L241 analogous -> reflected by

L243-245: since the overall p-value was > 0.05, you are discussing trends

L259-260 in terrestrial environments with higher salinity if they

L261: unique -> different

L291: of the P m strain to salinity

L265: 70 Sa (ref)

L285: demonstates -> suggests

L291 : natural abiotic environmental stressors -> additional abiotic stressors

L293-294: I don’t understand this statement. If strains have been selected to reflect a taxonomic aspect, it would be good to mention this at the end of the introduction or beginning of the methodology

L297:  P strains responded differently

 

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

All my comments have been taken into account.