Glycinebetaine Enhances Osmotic Adjustment of Ryegrass under Cold Temperatures
Round 1
Reviewer 1 Report
The manuscript entitled “Glycinebetaine enhances osmotic adjustment of ryegrass under cold temperatures” is devoted to an interesting topic. The study is scientifically robust, the data set extensive, and likely important for the general understanding of abiotic stress tolerance in plants. The objective of this paper is to determine the effects of exogenous GB application on growth of two ryegrass species under cold temperatures; however, most of the results and discussion is focused on other stress factors (like salinity, water and heat stress). Also, authors concluded that there is no evidence of GB natural accumulation in response to cold temperatures, while the ability to take up exogenously applied GB (evidenced as higher GB concentrations in leaves and stems) contributed to decreased osmotic potential, even though the effect of GB on growth was variable across the experiments. The originality of the work is not questionable. However, some serious doubts should be clarified.
Introduction. The introduction is short and concise. However, some facts about GB roles in abiotic stress tolerance in plants should be added to ensure an easier following the text to a broader audience. Recently published review article could be introduced (https://doi.org/10.3389/fpls.2019.00230). The objectives are clear, although they do not fully correspond to the title of the manuscript.
Material and Methods. The study itself consists of five different experiments. However, the connection among them is rather difficult to follow since they are different in the duration of the experiment, growing conditions, abiotic stresses, and GB concentrations applied exogenously. Were the photoperiod and light intensity the same in all experiments, as explained in experiment 1? For the quantification of changes in GB synthesis in response to various stresses (cold, heat, drought, salinity) plants were maintained in growth room for 21 days (experiment 1), while to quantify GB accumulation in ryegrass growing under different temperatures plants were maintained in growth room for only ten days (experiment 2). Similar, in experiments related to exogenous GB application (3, 4 and 5) plants were grown in a chamber for twelve weeks (to determine if the ryegrass species can take up exogenously applied GB and its effect on water relations) or two weeks (to see the effect of exogenously applied GB on growth under cold temperatures). Why were the plants of the same age not used for the different treatment applications? Furthermore, given the different duration of the experiments, were the plants in these experiments of the same age / developing stage when harvested for quantification of GB and water relation measurements (RWC and osmotic potential)? Which leaves were sampled for the analyses (first, second, all of them)? This crucial information is missing, as GB is known that exogenously applied it reallocates to actively growing and expanding tissues. As it is shown previously that synthesis of GB is also temporally delayed it would be very interesting and useful to see the proline status, if these measurements were made, since proline is also important as osmolyte. GB, similar like other compatible solutes, is suggested to be involved in ROS scavenging, so the lipid peroxidation level or ROS status could elucidate variable effect of GB on growth and provide more meaningful conclusions.
Results and Discussion. According to the objective pointed out in the introduction, the main results dealing with GB and cold stress are expected to be discussed. However, there are too many references related to the results obtained from experiments dealing with GB and drought, salinity and heat, but not so much related to the cold stress as the primary objective. So, the discussion should be rewritten appropriately.
Conclusions. The authors pointed out the capacity of investigated ryegrass species to take up exogenously applied GB in response to cold temperatures and concluded that higher concentrations of GB contribute to a decrease in osmotic potential (in agreement with the manuscript title), while the effect on growth was variable across the experiments. The effect of exogenous GB application on the growth and osmotic potential of plants at low temperatures could be better explained if the authors could provide results for proline content, lipid peroxidation levels, or the status of some ROS scavengers. The conclusions could then increase the scientific input.
The reference list is up to date and follows the format prescribed by the journal.
Minor editorial errors:
Line 159-160: the number 7 should be replaced with the number 0 in the sentence “QAC concentrations at 7, 15, and 22 days after…”
Line 171: footnotes of Table 1; no letters are describing significant differences among values
Line 178-179: a closing parenthesis is missing at the end of the sentence “This difference in effects on growth…. when GB was applied (Figures 3 and 4.
Line 217: Inside Table 3 ℃ is missing in the first column
Author Response
Reviewer 1
Introduction. The introduction is short and concise. However, some facts about GB roles in abiotic stress tolerance in plants should be added to ensure an easier following the text to a broader audience. Recently published review article could be introduced (https://doi.org/10.3389/fpls.2019.00230).
We added the following sentence to the paragraph that describes the positive effects of GB on stress tolerance:
Glycinebetaine plays a role in plant stress tolerance via protection of proteins and membranes and as an osmoticum that acts to lower cell osmotic potential under various osmotic stresses [7].
The reference cited is the review from Frontiers that the reviewer mentioned.
The objectives are clear, although they do not fully correspond to the title of the manuscript.
We are not sure how the objectives are in conflict with the title. We undertook this set of experiments to determine if GB would have a positive effect on growth under cold conditions and whether it would allow for osmotic adjustment as it does in other species. We did not see a consistent growth affect despite the consistent osmotic adjustment. We tried to capture this main result in the title of the paper.
Material and Methods. The study itself consists of five different experiments. However, the connection among them is rather difficult to follow since they are different in the duration of the experiment, growing conditions, abiotic stresses, and GB concentrations applied exogenously. Were the photoperiod and light intensity the same in all experiments, as explained in experiment 1?
To make this clearer, we changed “Light levels in this chamber and subsequent environments…” to “Light levels in this chamber and subsequent experiments…”
For the quantification of changes in GB synthesis in response to various stresses (cold, heat, drought, salinity) plants were maintained in growth room for 21 days (experiment 1), while to quantify GB accumulation in ryegrass growing under different temperatures plants were maintained in growth room for only ten days (experiment 2). Similar, in experiments related to exogenous GB application (3, 4 and 5) plants were grown in a chamber for twelve weeks (to determine if the ryegrass species can take up exogenously applied GB and its effect on water relations) or two weeks (to see the effect of exogenously applied GB on growth under cold temperatures). Why were the plants of the same age not used for the different treatment applications?
We don’t feel that the time differences are that great among the experiments. For the quantification of accumulation, plants were grown for 21+27=48 (expt 1) or 10+22=32 (expt 2) days. In both cases, these would be considered “young” plants. Experiment 3 was designed to determine the longer-term effects of exogenous GB application on GB accumulation and growth, whereas experiments 4 and 5 were designed to test the effects of exogenous GB application on growth of young plants. Each experiment was designed to address a slightly different question, and the length of time for growth was chosen to address that specific question. Rather than a negative, we believe the fact that we see consistent results in GB uptake and contribution to osmotic adjustment in plants of varying ages confirms the robust effects of GB in ryegrass.
Furthermore, given the different duration of the experiments, were the plants in these experiments of the same age / developing stage when harvested for quantification of GB and water relation measurements (RWC and osmotic potential)?
The plants were not the same age, but as we state above, we do not think this is critical to the interpretation of the results. We were not interested in replicating the effects on the same aged plants multiple times, but rather interested in the effects of GB in plants that once again would all be considered to be relatively young (as opposed to older, established field plants).
Which leaves were sampled for the analyses (first, second, all of them)? This crucial information is missing, as GB is known that exogenously applied it reallocates to actively growing and expanding tissues.
In all cases, all tissue was collected and a random subsample was used for GB quantification, as stated in section 2.3. Indeed, we understand that different leaf ages may accumulate different amounts of GB due to different uptake and/or translocation. However, our goal was to determine overall plant concentrations as opposed to spatial variability within the plant.
As it is shown previously that synthesis of GB is also temporally delayed it would be very interesting and useful to see the proline status, if these measurements were made, since proline is also important as osmolyte.
This is a good point and we agree. However, we did not quantify proline, or any other compounds that could act as potential osmolytes under the various stresses imposed. We did, however, see consistent accumulation of GB.
GB, similar like other compatible solutes, is suggested to be involved in ROS scavenging, so the lipid peroxidation level or ROS status could elucidate variable effect of GB on growth and provide more meaningful conclusions.
Again, we agree with this statement, but we did not measure ROS status.
Results and Discussion. According to the objective pointed out in the introduction, the main results dealing with GB and cold stress are expected to be discussed. However, there are too many references related to the results obtained from experiments dealing with GB and drought, salinity and heat, but not so much related to the cold stress as the primary objective. So, the discussion should be rewritten appropriately.
We appreciate this comment, but the fact is that there is very little in the literature on any effects of GB on plant growth under the specific conditions that we were testing. Again, our goal was not to test the potential role of GB on freeze tolerance of plant tissue. Very specifically, we set out to determine if GB could be an avenue to enhance growth of ryegrass species in the specific conditions of sub-optimal growth conditions. If this were the case, it has very large implications on pasture growth in many parts of the world. All that to say there is very little in the literature around this specific area. In response to this comment, we did another literature search and cannot find any further publications that would help us to interpret our results any more than we have to this point. If the goals of our work are still unclear, we are happy to re-write the introduction to do so.
Conclusions. The authors pointed out the capacity of investigated ryegrass species to take up exogenously applied GB in response to cold temperatures and concluded that higher concentrations of GB contribute to a decrease in osmotic potential (in agreement with the manuscript title), while the effect on growth was variable across the experiments. The effect of exogenous GB application on the growth and osmotic potential of plants at low temperatures could be better explained if the authors could provide results for proline content, lipid peroxidation levels, or the status of some ROS scavengers. The conclusions could then increase the scientific input.
We agree that all of this information would be interesting. However, we did not collect this data and do not have any material remaining from these experiments from which to obtain this data. The focus of our experiments was to assess the potential contribution of glycinebetaine to growth of rye grass under cold, not freezing (i.e. suboptimal, but not severely harmful) conditions, and therefore, we focused on how GB as an osmoticum might enhance growth. We understand that the reviewer may think our focus was too narrow, but we hope the paper still stands as a worthy set of experiments on the effects of GB on ryegrass growth under moderately cold conditions.
The reference list is up to date and follows the format prescribed by the journal.
Minor editorial errors:
Line 159-160: the number 7 should be replaced with the number 0 in the sentence “QAC concentrations at 7, 15, and 22 days after…”
Line 171: footnotes of Table 1; no letters are describing significant differences among values
Line 178-179: a closing parenthesis is missing at the end of the sentence “This difference in effects on growth…. when GB was applied (Figures 3 and 4.
Line 217: Inside Table 3 ℃ is missing in the first column
Thank you very much for these edits. All of these have been fixed.
Reviewer 2 Report
Reviewer’s comments
This paper is well written and interesting, and reports on studies of the effects of the osmo-protectant glycine betaine (GB) on annual (Lolium multiflorum) and perennial (L. perenne) ryegrass. The authors have under controlled conditions tested the effects of differing abiotic stresses on the accumulation of GB, but the main part of the study is on how exogenously applied GB affects accumulation of GB in the ryegrasses, and which effects it has on growth under cold temperatures. This is an interesting field since GB could alleviate stresses of pasture plants to e.g. cold.
Broad comments
As stated above, the paper is very well written and well structured. Still, there is some room for improvements.
The introduction is quite short and to the point, yet I would like to have a bit more information on how GB functions in plants – see e.g. the review by Annunziata et al 2019. Spatial and Temporal Profile of Glycine Betaine Accumulation in Plants Under Abiotic Stresses. Frontiers in Plant Science. DOI: 10.3389/fpls.2019.00230
In Materials and Methods, you should state the age of the plant when they went into the different experiments. The section on statistical analyses is very short. It should have more detailed information on which kind of models and effects were used for testing in the different experiments. In e.g. the growth analyses you tested the slopes of growth of GB treated plants against slope of control plants. What models did you use? You also stated that you compared mean values with Tukey’s multiple range test, however, in the figures and tables you have stated the use of several different tests. Please give some more details about this.
In the discussion I think you could start short with your own main findings, and then start to discuss the details.
Specific comments
L68 you describe the light conditions – were they the same for all the experiments?
L 75 You state that for each genotype x treatment you had three replicate containers. I suppose you meant species and not genotype, since you were working with seedlings of the two species and not clones. You need to correct this throughout the manuscript.
L 117 – Non-saturating light … ? Can you be more specific.
L141 replace “annual” with perennial
L145 – Figure text – please use the term Glycine betaine (GB) throughout, and not “quaternary ammonium compound”, since this is a bit confusing.
L 171 I am a bit confused with the statistics in Table 1. You have indicated significant different values with stars (not letters as described below the table), is this based on both species or just within species? You did not indicate significant difference in the osmotic potential of L. multiflorum, only effect on osmotic potential at full turgor, while in the text you state significant difference.
L 215 Table 3. You must go through the statistics and the indication of means that are significantly different. You have indicated significant differences e.g. of RWC at 18 oC between GB applications, but nothing is indicated after the values in the table. Likewise, for osmotic potential at full turgor. And on the other hand, you have indicated significant differences at 6 oC concerning succulence, where there is no significance.
L220 - ..then half of the plants were moved
L229 – replace…and cereals such as wheat..
L 230 – You have mixed the species – replace L. multiflorum with L. perenne. – You may have to rewrite the whole sentence since the logic of the whole sentence must change.
L 245 … Several field trials show no beneficial effects of ??? growth….
L 274 -277 – You are mixing the cv names – what is correct - L. perenne ´Kingston or Crusader? The growth was enhanced in L. perenne according to table 2.
L 278 Incomplete sentence
Comments for author File: 
Comments.docx
Author Response
Reviewer 2
This paper is well written and interesting, and reports on studies of the effects of the osmo-protectant glycine betaine (GB) on annual (Lolium multiflorum) and perennial (L. perenne) ryegrass. The authors have under controlled conditions tested the effects of differing abiotic stresses on the accumulation of GB, but the main part of the study is on how exogenously applied GB affects accumulation of GB in the ryegrasses, and which effects it has on growth under cold temperatures. This is an interesting field since GB could alleviate stresses of pasture plants to e.g. cold.
Broad comments
As stated above, the paper is very well written and well structured. Still, there is some room for improvements.
The introduction is quite short and to the point, yet I would like to have a bit more information on how GB functions in plants – see e.g. the review by Annunziata et al 2019. Spatial and Temporal Profile of Glycine Betaine Accumulation in Plants Under Abiotic Stresses. Frontiers in Plant Science. DOI: 10.3389/fpls.2019.00230
The paper that the reviewer mentions does a nice job of describing the described roles of GB in plants and indeed is a valuable reference for our introduction. Because of the scope of that paper, rather than provide a lengthy discussion of the potential roles of GB in plants, we added that reference and added the following sentence to the introduction:
Glycinebetaine plays a role in plant stress tolerance via protection of proteins and membranes and as an osmoticum that acts to lower cell osmotic potential under various osmotic stresses [7].
In Materials and Methods, you should state the age of the plant when they went into the different experiments. The section on statistical analyses is very short. It should have more detailed information on which kind of models and effects were used for testing in the different experiments. In e.g. the growth analyses you tested the slopes of growth of GB treated plants against slope of control plants. What models did you use? You also stated that you compared mean values with Tukey’s multiple range test, however, in the figures and tables you have stated the use of several different tests. Please give some more details about this.
In the discussion I think you could start short with your own main findings, and then start to discuss the details.
Specific comments
L68 you describe the light conditions – were they the same for all the experiments?
We are aware that light levels will be slightly different in every chamber and certainly over time. We regularly measured light levels and they were approximately 200 µmol m-2 sec-2 throughout all experiments. We added wording to this section to make this clearer.
L 75 You state that for each genotype x treatment you had three replicate containers. I suppose you meant species and not genotype, since you were working with seedlings of the two species and not clones. You need to correct this throughout the manuscript.
Yes, we were using the term “genotype” when referring to species, but the reviewer is correct that species is the more accurate and appropriate term. We changed throughout the manuscript.
L 117 – Non-saturating light … ? Can you be more specific.
We simply meant a low light level, but we understand that this term may be confusing so we removed it.
L141 replace “annual” with perennial
We now simply refer to L. perenne ‘Kingston’.
L145 – Figure text – please use the term Glycine betaine (GB) throughout, and not “quaternary ammonium compound”, since this is a bit confusing.
We made these changes.
L 171 I am a bit confused with the statistics in Table 1. You have indicated significant different values with stars (not letters as described below the table), is this based on both species or just within species?
Reviewer 1 also noted that we refer to letters when this is clearly not appropriate. This was simply a typo and it was removed. The significant differences indicated are within a species, although the main (GB application and species) and interaction (GB*Species) effects are presented below the means. We changed the statistical description from “within a column” to “within a species” to clarify this.
You did not indicate significant difference in the osmotic potential of L. multiflorum, only effect on osmotic potential at full turgor, while in the text you state significant difference.
We were considering the osmotic potential at full turgor to be a better indicator of osmotic adjustment, which is why we worded the results that way. To make things clearer, we re-worked that sentence to this:
Both species accumulated GB, which was a significant contributor to osmotic adjustment based on Yπ100 values (Table 1).
L 215 Table 3. You must go through the statistics and the indication of means that are significantly different. You have indicated significant differences e.g. of RWC at 18 oC between GB applications, but nothing is indicated after the values in the table. Likewise, for osmotic potential at full turgor. And on the other hand, you have indicated significant differences at 6 oC concerning succulence, where there is no significance.
The explanation for these supposed discrepancies has to do with the fact that we are presenting P values for overall main and interaction effects from the ANOVA at the bottom of the table, but the means presented are treatment levels within each species and temperature. In the case of the RWC at 18ï‚°C, the overall GB effect is significant when tested over both species, but within each species, this effect is not significant. The same is true in reverse for succulence at 6ï‚°C: the overall effects of GB (i.e. across both species) was ns, but within only L. multiflorum, it was significant. To try to make this clearer, we added the phrase “within a species x temperature level” to the description of the mean separation analysis. We hope this clarifies the two analyses that were done on this data.
L220 - ..then half of the plants were moved
Thank you for this suggestion—we made this change.
L229 – replace…and cereals such as wheat.
We made this change.
L 230 – You have mixed the species – replace L. multiflorum with L. perenne. – You may have to rewrite the whole sentence since the logic of the whole sentence must change.
Indeed, we made a mistake here. We have corrected this.
L 245 … Several field trials show no beneficial effects of ??? growth….
We added “of GB”.
L 274 -277 – You are mixing the cv names – what is correct - L. perenne ´Kingston or Crusader? The growth was enhanced in L. perenne according to table 2.
Thank you for picking this up. We have gone through the entire manuscript to make sure these errors were corrected.
L 278 Incomplete sentence
This was simply a typo. We removed this incomplete sentence.
Round 2
Reviewer 1 Report
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