Analysis of Cold-Developed vs. Cold-Acclimated Leaves Reveals Various Strategies of Cold Acclimation of Field Pea Cultivars
Round 1
Reviewer 1 Report
This is an interesting paper that presented the impact of the cold environment to the photosystem and growth of pea cultivars. The results were clearly presented and well discussed. However, in plant preparation, there is the main issue. Why don't you reserve some plants at 21 Celsius for control measurement? Thus, for the measurement in this study, the data were collected at different growth stages/ growing environment.
Another issue is that, what is the so-called "true leaf" cultivar? The authors should clearly clarify the "true leaf" definition.
Thirdly, the authors mentioned that the growth rate (RGR) of cultivar supposed to overcome the most severe winters decreased to the lowest values after the shift to 5°C. The reason, especially the mechanism, is not well discussed, even the authors indicated that the plant may store energy for the reproduction.
Thus, I would suggest the authors make some revision before it can be published.
Author Response
Comments and Suggestions for Authors
This is an interesting paper that presented the impact of the cold environment to the photosystem and growth of pea cultivars. The results were clearly presented and well discussed. However, in plant preparation, there is the main issue. Why don't you reserve some plants at 21 Celsius for control measurement? Thus, for the measurement in this study, the data were collected at different growth stages/ growing environment.
It was not our goal to compare the measured parameters during the cold stress with the parameters measured with plants kept in 21°C. The goal was to find out, what happens with the cultivars when they face to 5°C and to compare their response to this temperature treatment. Of course, the responses were different. Moreover, some of the responses also differed from what one would expect at first. One would expect that if the growth rate and photosynthetic parameters of given cultivars(s) are high or even higher at 5°C than at 21°C, these cultivars would be cold tolerant and would survive winter. However, opposite is true, since only ARK is known to be cold tolerant cultivar and this cultivar is characterized by decreased growth rate and decreased photosynthetic performance during the cold treatment. Thus, afila type cultivar with a behaviour similar to ARK, i.e. END cultivar, seems to be the most perspective for further search of the afila type cultivar useful for autumn sowing.
Another issue is that, what is the so-called "true leaf" cultivar? The authors should clearly clarify the "true leaf" definition.
We thank the reviewer for their valuable commentary. It is true that even stipules can be seen from physiological point of view as "true leaves." We meant this difference in rather morphological meaning. To clarify the difference we changed the term for Arkta to "normal-leaf" cultivar. This describe difference in morphology of ARK and another cultivars that instead of leaves develop stipules in form of tendrils. Term normal leaves refer to common leaves of pea plants as can be seen in old cultivars or wild-type plants of Pisum sativum.
Thirdly, the authors mentioned that the growth rate (RGR) of cultivar supposed to overcome the most severe winters decreased to the lowest values after the shift to 5°C. The reason, especially the mechanism, is not well discussed, even the authors indicated that the plant may store energy for the reproduction. Thus, I would suggest the authors make some revision before it can be published.
With respect to the mechanism, we have added following text to the discussion: This behavior is in agreement with literature; the cold acclimation is accompanied by decrease of growth (reviewed in [38]) and since sugars are not necessary to be synthetized to support the growth, starch, as a source of energy for further usage, is synthetized instead of sugars. Indeed, a negative correlation between plant growth and starch accumulation in plant leaves has been reported (reviewed in [39]). Further, the decreased sugar synthesis is accompanied by downregulation of photosynthetic activity, as observed in ARK (Figure 3 and 4), the downregulation being driven by reprogramming of gene expression (reviewed in [40]).
Author Response File: 
Author Response.docx
Reviewer 2 Report
This paper is interesting. The manuscript and figures are well showed. The results are in accordance with the goal of the study. The findings of this research are also interesting using remote sensing strategies for detecting fast potential germplasm for pea breeding to cold tolerance.
However, minor typographic mistakes throughout the manuscript should be checked and corrected (see attached file). Additionally, I recommend to be precise mentioning the aim of the study both in the abstract and the introduction.
in my opinion, after all these suggestions the manuscript should be accepted for publication.
Comments for author File: 
Comments.pdf
Author Response
Comments and Suggestions for Authors
This paper is interesting. The manuscript and figures are well showed. The results are in accordance with the goal of the study. The findings of this research are also interesting using remote sensing strategies for detecting fast potential germplasm for pea breeding to cold tolerance.
However, minor typographic mistakes throughout the manuscript should be checked and corrected (see attached file). Additionally, I recommend to be precise mentioning the aim of the study both in the abstract and the introduction.
The mistakes were corrected and the aims were added into the abstract and end of the introduction. We thank the reviewer for his time and an attempt to improve our manuscript.
In my opinion, after all these suggestions the manuscript should be accepted for publication.
peer-review-5553710.v1.pdf
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript “Analysis of cold-developed vs. cold-acclimated leaves reveals various strategies of cold acclimation of field pea cultivars” by Huŝicková et al. presents a comparison of four pea cultivars grown in 5 °C cold treatment of 22 days. The methods include chlorophyll fluorescence imaging as well as OJIP curves and chlorophyll content. The experimental setup does not have a control temperature to compare with, but instead the results of the 22-day-cold treatment are compared to the day 0 before the treatment started. Therefore, the results allow comparison of the four cultivars and development of cold response, but not comparison to control plants of the same age.
My questions:
The title states that Analysis of cold-developed vs. cold-acclimated leaves reveals various strategies of cold acclimation of field pea cultivars. Which strategies are these? Could these be clearly described as differences between cold-developed and cold-acclimated leaves of the different cultivars? The abstract lacks the word cold-acclimated whereas the title gives the reader an expectation of comparison of the two leaf types.
What is the use of the results for those that are not interested in these specific pea cultivars? Can one conclude anything about the cold acclimation process on the basis of these results?
Is +5 °C for 22 days a cold treatment? Or is it only a chilling treatment? Could cold treatment refer to freezing temperatures?
Abstract L 24-26. “Arkta proved the fastest retardation of photosynthesis and shoot growth, but it recovered when there did not occur further temperature decreases. “ Do you know what happens in Arkta with further decrease in temperature? The sentence leads to an impression that the manuscript describes a further decrease in temperature as well, which does not occur.
The manuscript would benefit from English language correction. There are many strange word choices or sentences that are difficult to grasp. Here some examples but not a comprehensive list.
L44 impulses –> signals?
L46 Cold acclimation occurs in species from temperate regions when the temperature decreases to and sustain for some time at the low but still non-freezing degree. -> the temperature decreases to a low but still non-freezing temperature and stays low for a period of time?
L56 agriculturalists –> farmers or farming?
L402 All over -> To conclude / At the end
L418 perspective -> promising / potential
Materials and methods
The words cold-acclimated and cold-developed are not explained in the materials and methods. The definition of these leaf types is needed. Moreover, their age difference and description of possible size differences is needed.
Figure 2 describes the leaves that were taken as samples as being 2nd leaves/stipules (cold-acclimated) or on 3rd leaves/stipules, but this is not described in Materials and Methods. Which leaves or stipules were the ones taken as samples before the start of the experiment (on day 0)?
The definitions of the cold resistance of the four cultivars tested should be described in Materials and methods for each cultivar. Now the knowledge that Arkta, Enduro and CDC Le Roy are cold resistant and that Terno is sensitive to cold comes in the results section and it remains obscure to the reader whether this is previous well documented knowledge or something that is concluded somehow from the results of the present study. If this is previous knowledge, then the source (reference) for the knowledge should be stated.
Fig 1 legend. Which leaves were measured? Did it differ from one day to a next one? For the figure legend to stand alone, the statistical test and p<0.05 should be mentioned.
Fig 2. I totally dislike the word “control” since it implies that the control, cold-acclimated and cold-developed leaves were sampled the same day from different plants, which was not the case. I would rather use labels “day 0” and “day 22”, the latter with cold-acclimated and cold developed leaves. For the figure legend to stand alone, the pea cultivars should be mentioned.
Fig 3 legend. The pea cultivars and the course of cold treatment should be mentioned. What does leaf level mean?
Fig 4 legend. The pea cultivars, which leaves or stipules?
Fig 5. The numbers along the colour code are not readable. What do the colours mean?
The results and discussion include a lot of detailed description of the different responses of the cultivars? Do these results remain descriptive or can you conclude something that would benefit someone else but the farmer that thinks of whether to plant END or CDC?
The discussion would benefit from less repetition of the results and more interpretation of the wider picture. Major findings should be told in the first chapter of the discussion to give them more emphasis.
350-351. What does this mean? “Nevertheless the cold-acclimating stipules of the TER, END and CDC exhibited opposite trend than the predominantly observed.”
L376 Such response -> no response?
L395 “although, contrary to ARK, this inhibition manifests predominantly on the cold-developed stipules” -difficult to grasp as such, since ARK does not have stipules and thus contrary to ARK could mean in stipules instead of leaves, even though I assume that that was not meant.
L411-413 “we conclude that END and CDC do not have characteristics of a winter cultivar suitable for winters of European central zone.” is in conflict with L417- 418 “Thus, from all the tested pea cultivars, END seems to be the most perspective for further search of the afila type cultivar useful for autumn sowing.”
Author Response
Comments and Suggestions for Authors
The experimental setup does not have a control temperature to compare with, but instead the results of the 22-day-cold treatment are compared to the day 0 before the treatment started. Therefore, the results allow comparison of the four cultivars and development of cold response, but not comparison to control plants of the same age.
We are very grateful to the reviewer for his conscientious work on the review of our manuscript. We believe the reviewer helped substantially to enhance the quality of the text.
My questions:
The title states that Analysis of cold-developed vs. cold-acclimated leaves reveals various strategies of cold acclimation of field pea cultivars. Which strategies are these? Could these be clearly described as differences between cold-developed and cold-acclimated leaves of the different cultivars? The abstract lacks the word cold-acclimated whereas the title gives the reader an expectation of comparison of the two leaf types.
To answer and satisfy these reviewer questions, the abstract has been completely rewritten.
What is the use of the results for those that are not interested in these specific pea cultivars? Can one conclude anything about the cold acclimation process on the basis of these results?
Yes we believe there is a message that have wider significance: the conclusion about the alertness concerning the evaluation of parameters from whole plants, and attention to organs which developed under the stress, which is stated in the conclusion as well as in the abstract.
Is +5 °C for 22 days a cold treatment? Or is it only a chilling treatment? Could cold treatment refer to freezing temperatures?
In general there cannot be made conclusions about the freeze-tolerance on the basis of pure cold treatment. In our study, however, we tested a cultivar that is well known for its abilities to survive the freezing winters of European central zone. On the basis of this knowledge and on the basis of comparison of this cultivar with other 3 cultivars we searched for parameters that could help to find traits signalling the potential for breeding of a new cold-resistant cultivar. At the same time we paid attention not to generalize our conclusions from cold treatment to freeze-tolerance.
Abstract L 24-26. “Arkta proved the fastest retardation of photosynthesis and shoot growth, but it recovered when there did not occur further temperature decreases. “ Do you know what happens in Arkta with further decrease in temperature? The sentence leads to an impression that the manuscript describes a further decrease in temperature as well, which does not occur.
The sentence has been changed. The freeze-survival of Arkta is well described in [28] where it is regarded as a benchmark for tests of winter survival.
The manuscript would benefit from English language correction. There are many strange word choices or sentences that are difficult to grasp. Here some examples but not a comprehensive list.
L44 impulses –> signals?
L46 Cold acclimation occurs in species from temperate regions when the temperature decreases to and sustain for some time at the low but still non-freezing degree. -> the temperature decreases to a low but still non-freezing temperature and stays low for a period of time?
L56 agriculturalists –> farmers or farming?
L402 All over -> To conclude / At the end
L418 perspective -> promising / potential
The text has been edited by a professional native English speaking editor and several parts of the text (including all the sentence mentioned by the reviewer) have been rewritten into a more intelligible form.
Materials and methods
The words cold-acclimated and cold-developed are not explained in the materials and methods. The definition of these leaf types is needed. Moreover, their age difference and description of possible size differences is needed.
The explanation of the words “cold-acclimated” and “cold-developed” has been added into the text of Materials and Methods as well as into the abstract. By the words "cold-acclimated" and "cold-developed" leaves/stipules we want to emphasize the difference of conditions that influenced development of particular leaves/stipules. Cold-acclimated leaves/stipules finished their development before cold treatment, to which they acclimate. Cold-developed leaves have grown and developed under cold-treatment. We evidenced in this study that leaves developed in optimal conditions behaves differently from leaves that underwent development in cold.
To describe developmental shifts in the cultivar we re-analyse our RGB images to discover number of plant with fully unfolded (photosynthetically active) leaves. We counted plants with fully opened 3rd leaves in each time point during cold-treatment. Whereas ARK opened 3rd leaves at the beginning of cold-treatment, other cultivars opened 3rd leaves later but gradually during entire time of cold treatment. This suggest that ARK opened 3 leaves as response to previous optimal conditions but then blocked its development (as seen also from RGR data), but other cultivars do not respond to cold so dramatically and continued in development even under suboptimal conditions. This information have been added to the manuscript as supplementary file S1.
Figure 2 describes the leaves that were taken as samples as being 2nd leaves/stipules (cold-acclimated) or on 3rd leaves/stipules, but this is not described in Materials and Methods. Which leaves or stipules were the ones taken as samples before the start of the experiment (on day 0)?
The required data were added into the figure legend and into the chapter of Materials and Methods.
The definitions of the cold resistance of the four cultivars tested should be described in Materials and methods for each cultivar. Now the knowledge that Arkta, Enduro and CDC Le Roy are cold resistant and that Terno is sensitive to cold comes in the results section and it remains obscure to the reader whether this is previous well documented knowledge or something that is concluded somehow from the results of the present study. If this is previous knowledge, then the source (reference) for the knowledge should be stated.
This knowledge connected with the references had been stated in the text of Introduction, but we agree with the reviewer that it is important to be mentioned in the section of Materials and Methods as well. Cold resistance of tested cultivars together with the related references has been thus added into the Table 1.
Fig 1 legend. Which leaves were measured? Did it differ from one day to a next one? For the figure legend to stand alone, the statistical test and p<0.05 should be mentioned.
All the required data were added into the figure legend. The information concerning the statistical tests was added into each figure legend were applicable.
Fig 2. I totally dislike the word “control” since it implies that the control, cold-acclimated and cold-developed leaves were sampled the same day from different plants, which was not the case. I would rather use labels “day 0” and “day 22”, the latter with cold-acclimated and cold developed leaves. For the figure legend to stand alone, the pea cultivars should be mentioned.
The word “control” in the described sense has been replaced throw all the text as well as in the figure.
Fig 3 legend. The pea cultivars and the course of cold treatment should be mentioned. What does leaf level mean?
All the questioned data were added into the figure legend. The expression “leaf level” does have no sense in this text anymore and thus was removed. The list of pea cultivars was added to each figure legend.
Fig 4 legend. The pea cultivars, which leaves or stipules?
All the required data were added into the figure legend.
Fig 5. The numbers along the colour code are not readable. What do the colours mean?
The size of numbers on the axes have been increased. The information concerning the meaning of colours has been added.
The results and discussion include a lot of detailed description of the different responses of the cultivars? Do these results remain descriptive or can you conclude something that would benefit someone else but the farmer that thinks of whether to plant END or CDC?
To the first question: One of the goals of the work was to find parameter(s), which is (are) suitable for identification of pea cultivars resilient to cold treatment. We believe description of changes of different parameters of different cultivars essentially belongs to the righteous selection.
To the second question: Yes we proposed parameters which are sensitive and valuable for further screening of plant tolerance and pointed out that the organs developed under stress might bring the important piece of information when evaluated separately.
The discussion would benefit from less repetition of the results and more interpretation of the wider picture. Major findings should be told in the first chapter of the discussion to give them more emphasis.
The discussion has been rewritten in the above mentioned sense.
350-351. What does this mean? “Nevertheless the cold-acclimating stipules of the TER, END and CDC exhibited opposite trend than the predominantly observed.”
We apologize for low comprehensibility of the sentence. Better expression might be: “Nevertheless the cold-acclimating stipules of the TER, END and CDC exhibited opposite trend than was observed on ARK and described in the literature.” As we mentioned above, the language of whole text has been edited to increase the intelligibility.
L376 Such response -> no response?
The sentence has been changed.
L395 “although, contrary to ARK, this inhibition manifests predominantly on the cold-developed stipules” -difficult to grasp as such, since ARK does not have stipules and thus contrary to ARK could mean in stipules instead of leaves, even though I assume that that was not meant.
To make the massage clear the sentence has been corrected.
L411-413 “we conclude that END and CDC do not have characteristics of a winter cultivar suitable for winters of European central zone.” is in conflict with L417- 418 “Thus, from all the tested pea cultivars, END seems to be the most perspective for further search of the afila type cultivar useful for autumn sowing.”
We believe both the sentences are valid. As we state in the discussion, despite the fact that END does not bear the characteristics of the winter cultivar suitable for European continental winters, it might be useful for further research and breeding oriented toward winter cultivars of afila type. But we agree with the reviewer that these sentences might sound contradictory. To reduce the notional discrepancy they have been modified.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
I am delighted to see the good improvement of the manuscript.
Minor comments:
"soluble sugars" instead of "sugars" would improve readability at places, for example on lines 393-395 ("since sugars are not necessary to be synthesized to support growth, starch, as a source of energy for further usage, is synthesized instead of sugars"), since starch is a sugar polymer
On lines 452-454 "Thus, in the afila cultivars the most promising results were exhibited by the END cultivar, which showed the highest sensitivity to cold, which can be seen as a storage preservation mechanism for overwintering." may be difficult to grasp- how about "the highest immediate response to cold" or maybe with a specification of which type of response
Author Response
Minor comments:
"soluble sugars" instead of "sugars" would improve readability at places, for example on lines 393-395 ("since sugars are not necessary to be synthesized to support growth, starch, as a source of energy for further usage, is synthesized instead of sugars"), since starch is a sugar polymer
Response: It is absolutely true. We apologize for such an ambiguity and thank the reviewer for their attention. The text concerning sugars has been changed into following:
“since sugars are not necessary to be synthesized to support growth, starch, as a source of energy for further usage, is synthesized preferentially at the expense of monosaccharides. Indeed, a negative correlation between plant growth and starch accumulation in plant leaves has been reported (reviewed in [39]). Further, these changes are accompanied by downregulation of photosynthetic activity, as observed in ARK …”
On lines 452-454 "Thus, in the afila cultivars the most promising results were exhibited by the END cultivar, which showed the highest sensitivity to cold, which can be seen as a storage preservation mechanism for overwintering." may be difficult to grasp- how about "the highest immediate response to cold" or maybe with a specification of which type of response
Response: Thus, in the afila cultivars the most promising results were exhibited by the END cultivar, which showed the highest response to cold in the form of photosynthesis and growth inhibition, which can be seen as a storage preservation mechanism for overwintering.
Author Response File: Author Response.docx
