Humidity and Light Modulate Oxygen-Induced Viability Loss in Dehydrated Haematococcus lacustris Cells
Round 1
Reviewer 1 Report
This is a very good paper, building on prior work by authors and collaborators on desiccation tolerance in Haematoccocus lacustris, herein assessing methods for best germplasm conservation/storage of this species by evaluating reasons for loss of viability under a range of storage environments. I have only a few minor comments which I suggest need addressing.
1. While it is clear that H. lacustris needs to be acclimated to achieve desiccation tolerance - this is based on previous work by the authors (ref 23). In the current paper, the authors refer to comparison of data with non-acclimated cells, stating data not shown. I feel it should be made clear to the reader at some point what the acclimation process requires and why it is necessary.
2. I found an apparent contradiction in lines 320-321 "After 3 years, there was a slight decrease in Fv/Fm values of rehydrated cells previously stored at sub-zero temperatures", to what is stated in line 294 which states “Ageing at these conditions was not significant over the 3 years..."
3. Minor typograpical errors can be found highlighted in yellow - as can comments about 1 and 2 above, in your article attached by myself.
After 3 years, there was a slight decrease in Fv/Fm values of rehydrated cells previously stored at sub-zero temperatures Line 320-321. This is in contradiction to the statement in lines 294 above which state “Ageing at these conditions was not significant over the 3 years.
Comments for author File: 
Comments.pdf
Author Response
This is a very good paper, building on prior work by authors and collaborators on desiccation tolerance in Haematoccocus lacustris, herein assessing methods for best germplasm conservation/storage of this species by evaluating reasons for loss of viability under a range of storage environments. I have only a few minor comments which I suggest need addressing.
RESPONSE: Thank-you for reviewing our submission. We are happy you were positive about its content!
- While it is clear that H. lacustris needs to be acclimated to achieve desiccation tolerance - this is based on previous work by the authors (ref 23). In the current paper, the authors refer to comparison of data with non-acclimated cells, stating data not shown. I feel it should be made clear to the reader at some point what the acclimation process requires and why it is necessary.
RESPONSE: Text was added in the introduction on lines 87-91: "The acclimation process of liquid-cultivated cells required atmospheric exposure for a few days in a humid atmosphere, and light to drive photosynthesis, but not necessarily high light stress [23]. Without acclimation, many liquid-grown cells collapsed during dehydration, while those that remained intact did not recover photosynthetic activity during rehydration, showing lack of desiccation tolerance."
- I found an apparent contradiction in lines 320-321 "After 3 years, there was a slight decrease in Fv/Fm values of rehydrated cells previously stored at sub-zero temperatures", to what is stated in line 294 which states “Ageing at these conditions was not significant over the 3 years..."
RESPONSE: We changed the sentence to "there was a slight, but insignificant, decreases in Fv/Fm" to lines 320-321
- Minor typograpical errors can be found highlighted in yellow - as can comments about 1 and 2 above, in your article attached by myself.
RESPONSE: We modified the text in the introduction referring to the first point highlighted in the pdf (see response above). The other typos were corrected.
After 3 years, there was a slight decrease in Fv/Fm values of rehydrated cells previously stored at sub-zero temperatures Line 320-321. This is in contradiction to the statement in lines 294 above which state “Ageing at these conditions was not significant over the 3 years.
RESPONSE: See our response above to this point.
Reviewer 2 Report
The manuscript entitled "Humidity and light modulate oxygen-induced viability loss in dehydrated Haematococcus lacustris cells" addresses a very relevant and appropriate topic for this journal.
The manuscript is well structured, well-written and well-founded. Some minor fixes are suggested below.
Corrections needed:
line 26 - Keywords: Haematococcus lacustris, desiccation tolerance, reactive oxygen species, antioxidants, ...
line 46 - 25 °C, below a threshold around 44-49% RH, the cytoplasm solidifies into a glassy state,
line 98 - ... and melt. Therefore, cells were equilibrated to 50% RH at 20 °C, which corresponded
line 257 - above and below these peaks (20 and −80 °C) and within the range of the lipid melting
line 270 - 3.2. Temperature dependency of ageing reactions in desiccated H. lacustris cells (note: "H. lacustris " in italics)
line 284 - storage at (b) 20 °C, (c) 4 °C, and (d) −20 °C, on a false colour scale shown below the images.
line 288 - for cells stored at 20 and 4 °C, respectively. Ageing at these conditions was significant
line 308 - both –50 and –80 °C, leading to a slope with smaller correlation coefficient, may be an ...
line 393 - rapidly fell as viability was lost (Fig. S4). In contrast, at 80% RH, γ-glutamyl-cysteine
Author Response
The manuscript entitled "Humidity and light modulate oxygen-induced viability loss in dehydrated Haematococcus lacustris cells" addresses a very relevant and appropriate topic for this journal.
The manuscript is well structured, well-written and well-founded. Some minor fixes are suggested below.
RESPONSE: Thank-you for reviewing our submission. We are happy you were positive about content!
Corrections needed:
line 26 - Keywords: Haematococcus lacustris, desiccation tolerance, reactive oxygen species, antioxidants, ...
RESPONSE: We added a semicolon between keywords
line 46 - 25 °C, below a threshold around 44-49% RH, the cytoplasm solidifies into a glassy state,
RESPONSE: We added 'and' between "25 °C" and "below a threshold around 44-49%"
line 98 - ... and melt. Therefore, cells were equilibrated to 50% RH at 20 °C, which corresponded
RESPONSE: The sentence was modified to "cells were equilibrated to 50% RH and 20°C, (conditions that lead the cytoplasmic aqueous phase to form a glassy state) before storage in the dark for up to three years at temperatures between −80 °C and 20 °C."
line 257 - above and below these peaks (20 and −80 °C) and within the range of the lipid melting
RESPONSE: The sentence was modified to: "Based on this thermal profile, temperatures above and below these peaks (20 °C and −80 °C, respectively), and within the range of the lipid melting peaks (4 °C, −20 °C and −50 °C), were used for long term storage of cells in Experiment 1."
line 270 - 3.2. Temperature dependency of ageing reactions in desiccated H. lacustris cells (note: "H. lacustris " in italics)
RESPONSE: Since the heading is in italics, the species name was intentionally left non italic.
line 284 - storage at (b) 20 °C, (c) 4 °C, and (d) −20 °C, on a false colour scale shown below the images.
RESPONSE: The text was changed to "Fv/Fm values from (a), but as images of individual cells using a false-colour scale (below), after 1 year in storage at (b) 20°C, (c) 4°C, and (d) −20°C."
line 288 - for cells stored at 20 and 4 °C, respectively. Ageing at these conditions was significant
RESPONSE: We changed the text to "Loss of Fv/Fm under these conditions was significant" and deleted the reference to years for clarity
line 308 - both –50 and –80 °C, leading to a slope with smaller correlation coefficient, may be an ...
RESPONSE: The sentence was changed to "Differences in slope coefficients due to the similar ageing rate at –50 °C and –80 °C may be an artefact of the calculations..."
line 393 - rapidly fell as viability was lost (Fig. S4). In contrast, at 80% RH, γ-glutamyl-cysteine
RESPONSE: The sentence was changed to "However, with longer time, and similar to GSH concentrations, γ-glutamyl-cysteine concentrations rapidly fell as an increasing number of cells lost viability"
