Memory Effects of Water Deprivation in European Beech (Fagus sylvatica L.) and Silver Fir (Abies alba Mill.) Seedlings Grown in Mixed Cultivation
, Michael Dannenmann 5, Xueying Quan 6, Daniel Maurer 2 and Heinz Rennenberg 2,7
Round 1
Reviewer 1 Report
General comments
I have read the manuscript (forests -1934123). Entitle: Memory effects of water deprivation in European beech (Fagus sylvatica L.) and white fir (Abies alba Mill.) seedlings grown in mixed cultivation written by Fengli Yang et. al., for publication of forests MDPI. In this study, the author investigated two popular forest tree species European beech and silver fir for mature forests for their drought tolerance and memory effect of the drought exposure grown in mixed cultivation. The results showed that previous-year drought hardening mediated Author found enhanced biomass accumulation (leaves and root) in the subsequent year in beech but not impact fir growth. Memory drought showed a significant effect on relative water contents, total N, and soluble protein contents in leaves and roots of beech and fir but resulted in decreased total amino acid contents. Moreover, the memory effect is stronger in beech than in fir.
The overall research is well conducted, and research is obvious application potential because the present study highlights that drought hardening induces memory effects. In this sense,e manuscript is much valuable. However, I found some points, especially the flow of the text and lack of potential references, and lack of connection of story in different paragraphs, especially in the introduction and discussion sections. The author should provide enough examples and their interpretation of different traits of physiological and biochemical responses by the latest and appropriate references, some of which I mentioned below. Overall after I evaluate and request the author for this manuscript as a “MAJOR REVISION”.
Major suggestions
1) Introduction: The introduction is well starting with the issue of climate change and its direct effect of forest dieback in central Europe which is highly appreciated. However, the important message in the introduction especially the effect of drought on forest species plants should be mentioned primarily in the initial part before entering the detailed literature review of the background. The article https://doi.org/10.1016/j.foreco.2020.118099 better presented the drought effect on the plant, please follow this article and cited, author should mainly mention that “drought reduced the morphological and physiological traits, reduce the photosynthesis, leaf water potential and sap movement and reduction of stomatal conductance.
2) Objectives of the research: The author presented a clear hypothesis from the Ln. 91-95 but here the author should include the research objectives as well. The author should be well connected to these two parts while mentioning the research objective. The research objective should be very clear in the introduction sections because, without appropriate literature, questions, or research objectives in the introduction section the entire text will be unclear.
3) Discussion: Author should Improve the discussion section more logically and concisely with potential references. Why is ROS emerging in stress conditions? Especially drought? The author should address it somewhere in the discussion. Refer to these two articles for better clarify (1) https://doi.org/10.1038/s41598-019-55889 (2) https://doi.org/10.1016/j.scitotenv.2021.146466 and mention somewhere in that paragraph “drought stress plant produces the ROS when these plant exposed to the drought and plant produce antioxidant, and secondary metabolites and those play to the role for protecting the plant for detoxifying ROS and protect the plant to protect the abnormal condition (i.e. stress) and also help protein and amino acid stabilization”.
Some other comments
4) Line no. 20: Author should be more careful at the beginning of any sentences, in Ln 20, starting of the sentences with “Only” is redundant, please rephrase this sentence. Moreover, the author should apply this suggestion throughout the manuscript. Moreover, the beginning of the words is bold, it should not be.
5) Line no. 112: The soil moisture sensor, “EC-5” should be out of the parenthesis, only instrumental detil and company detail should be kept inside the parenthesis. Please similarly follow in other used instrumental parts throughout the manuscript.
6) Line no. 128: The whole section 2.3 is not much clear, especially how the author created the drought stress and how to compare with the control plant. What is “mesocosms serving” ? I am not familiar with that. Also text is more confusing, could you please rewrite it.
7) Line 312 (Discussion): Author should concise the discussion by cutting of less important text and referring to the below articles and citing them and interpreting the carbon isotope based on the two study pant materials. Please deal with the δ13C. refer and cite drought, waterlogging, salinity related articles as references https://doi.org/10.1016/B978-0-12-816091-6.00004-3 https://doi.org/10.1016/j.agrformet.2022.109071 and https://doi.org/10.1111/nph.12423 of carbon isotopic discrimination (stable isotope/ δ13C) is the proxy of WUE and plant water relation and the useful method to insights of the chemical, physical and metabolic process involved in carbon transformation in the stressed plants.
8) Line no. 453 (Conclusion): The conclusion should not be repetitive in the abstract or a summary of the results section. I would love to read striking points and take-home messages that will linger in the readers’ minds. What is the novelty, how does the study elucidate some questions in this field, and the contributions the paper may offer to the scientific community?
9) Line no. 456 & 465: In the conclusion section, the literature citation is not good. Please remove this from the conclusion and adjust the literature in the other section or completely remove and mainly focus on the main message of your study and emphasis how your research provides benefit to others or the ecosystem.
10) Line no. 474 (Reference): please double-check the citations, their style, spell check, and other grammatical errors. moreover, I request to the authors for revision throughout the manuscript according to the journal rules.
Good Luck!
Author Response
Major suggestions
1) Introduction: The introduction is well starting with the issue of climate change and its direct effect of forest dieback in central Europe which is highly appreciated. However, the important message in the introduction especially the effect of drought on forest species plants should be mentioned primarily in the initial part before entering the detailed literature review of the background. The article https://doi.org/10.1016/j.foreco.2020.118099 better presented the drought effect on the plant, please follow this article and cited, author should mainly mention that “drought reduced the morphological and physiological traits, reduce the photosynthesis, leaf water potential and sap movement and reduction of stomatal conductance.
Response: This information is now provided in line 47-51. The publication of Bhusal et al. 2020 is cited here.
2) Objectives of the research: The author presented a clear hypothesis from the Ln. 91-95 but here the author should include the research objectives as well. The author should be well connected to these two parts while mentioning the research objective. The research objective should be very clear in the introduction sections because, without appropriate literature, questions, or research objectives in the introduction section the entire text will be unclear.
Response: This paragraph has been rephrased, please see line 82-87 and line 92-94. The research objective of the present study is given in line 92-94.
3) Discussion: Author should Improve the discussion section more logically and concisely with potential references. Why is ROS emerging in stress conditions? Especially drought? The author should address it somewhere in the discussion. Refer to these two articles for better clarify (1) https://doi.org/10.1038/s41598-019-55889 (2) https://doi.org/10.1016/j.scitotenv.2021.146466 and mention somewhere in that paragraph “drought stress plant produces the ROS when these plant exposed to the drought and plant produce antioxidant, and secondary metabolites and those play to the role for protecting the plant for detoxifying ROS and protect the plant to protect the abnormal condition (i.e. stress) and also help protein and amino acid stabilization”.
Response: ROS production and scavenging is explained in line 53-58 in the Introduction. Moreover, the two reference of Khaleghi et al. 2019 and Bhusal et al. 2021 are cited here.
Some other comments
4) Line no. 20: Author should be more careful at the beginning of any sentences, in Ln 20, starting of the sentences with “Only” is redundant, please rephrase this sentence. Moreover, the author should apply this suggestion throughout the manuscript. Moreover, the beginning of the words is bold, it should not be.
Response: “Only” has been replaced by “Very” without bold format. Please see line 20. We also checked the whole manuscript, there is no more such situation.
5) Line no. 112: The soil moisture sensor, “EC-5” should be out of the parenthesis, only instrumental detil and company detail should be kept inside the parenthesis. Please similarly follow in other used instrumental parts throughout the manuscript.
Response: Here and others in the text have been corrected. Please see line 111, and line 152-157, line 176.
6) Line no. 128: The whole section 2.3 is not much clear, especially how the author created the drought stress and how to compare with the control plant. What is “mesocosms serving” ? I am not familiar with that. Also text is more confusing, could you please rewrite it.
Response: This section has been rewritten, please see line 128-134.
7) Line 312 (Discussion): Author should concise the discussion by cutting of less important text and referring to the below articles and citing them and interpreting the carbon isotope based on the two study pant materials. Please deal with the δ13C. refer and cite drought, waterlogging, salinity related articles as references https://doi.org/10.1016/B978-0-12-816091-6.00004-3, https://doi.org/10.1016/j.agrformet.2022.109071 and https://doi.org/10.1111/nph.12423 of carbon isotopic discrimination (stable isotope/ δ13C) is the proxy of WUE and plant water relation and the useful method to insights of the chemical, physical and metabolic process involved in carbon transformation in the stressed plants.
Response: The discussion of carbon isotopic discrimination has been added in line 318-320. The three papers suggested are cited here. Please see line 319-323: “δ13C is an important integrative proxy of plant water use efficiency, water relations and stomatal movement, and is widely employed for interpreting chemical, physical and metabolic process involved in carbon transformation in stressed plants (Cernusak et al. 2013, Bhusal et al. 2022, More et al. 2022). In the present study, memory effects of drought on foliar δ13C were not observed, neither in beech leaves nor in fir needles, except for a minor variation in beech roots (Figs. S17, S18)”.
8) Line no. 453 (Conclusion): The conclusion should not be repetitive in the abstract or a summary of the results section. I would love to read striking points and take-home messages that will linger in the readers’ minds. What is the novelty, how does the study elucidate some questions in this field, and the contributions the paper may offer to the scientific community?
Response: The conclusion has been rephrased and several striking points have been given. The reference has been removed. Please see line 394-408.
9) Line no. 456 & 465: In the conclusion section, the literature citation is not good. Please remove this from the conclusion and adjust the literature in the other section or completely remove and mainly focus on the main message of your study and emphasis how your research provides benefit to others or the ecosystem.
Response: The references have been removed. Please see line 394-408.
10) Line no. 474 (Reference): please double-check the citations, their style, spell check, and other grammatical errors. moreover, I request to the authors for revision throughout the manuscript according to the journal rules.
Response: Have been rechecked and adapted the manuscript to the journal rules.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Interesting research!
Some general remarks:
- Maybe some figures can be put together? E.g. fig 2 and 3, Fig 1 and 4, fig 9, 10 and 11 etc…
- I wonder in how far the height of the individual seedlings (above ground) influenced their different N contents that were measured. Different statistical methods allow for accounting for the height of the seedlings while comparing drought treated and control groups.
- Personally, I would have liked to have seen p-values for the different statistical tests in the text (when mentioning that this or that is/was significantly different from another), but I understand it may make the text more difficult to read.
Some detailed remarks:
Line 82: trigged, maybe it should be triggered?
Line 101: The mean lengths of beech and fir seedlings at planting were 21 cm and 31 cm for shoot, 14 cm and 24 cm for root, respectively. How did you measure the “length” of the root? Also, give standard deviations.
Line 147: FW – DW)/FW; maybe explain what the abbreviations FW and DW stand for (fresh weight, dry weight I suppose)?
Line 188: Data shown are means + S.D. Aren’t the figures normal box plots?
Line 202: Significant differences between the number of beech neighbours are shown by different capital letters. I do not see this in the figure?
Line 208: It was also observed, when data were separated according to the number of neighbouring firs, but was not significantly affected by this number (Figs. 2, S2). Maybe rephrase?
Line 212: …a decrease in biomass at 5 neighbouring beech seedlings (Figs. 1, 3, S3). This is not so clear to me when I look at the figures.
Line 229: Is there no N in the stems? (idem for C)
Line 277: Total amino acid N of European beech and silver fir seedling… Maybe the N should be deleted? Or it should be added in the axis title of the figure and in the main text? Also in line 291
Line 298: In silver fir, structural N in current year needles was reduced, but not significantly affected in previous year needles and roots by drought hardening in the previous year. Highest structural N was recorded in previous year needles; lower but similar contents were found in current year needles and roots (Fig. 11). Maybe rephrase both sentences to make the results more clear?
Line 341: This result is consistent with the present result showing that drought altered carbon contents, observed directly after mild water deprivation and rewetting as mechanism of drought hardening (Yang et al. 2022), were largely reversed in the subsequent year (Fig. 4). Does fig 4 show this statement?
Line 344: Moreover, in contrast to the generally decreased root biomass observed in pure beech cultures upon drought (Davidson et al. 1992, Fotelli et al. 2001, Löf 345 et al. 2005, Meier and Leuschner 2008, Rose et al. 2009, Tomasella et al. 2019), the facilitation of fir on beech observed in the previous year (Yang et al. 2022) was also observed in the subsequent year of cultivation (Figs.1, 2, S1 ). “The facilitation of fir on beech “ is not so clear to me.
Line 366:.. phenotypic and morphological effects… Are these two not the same?
Line 378: …observed stimulated post-drought growth… There is also quite some literature on post-drought “compensation growth” in woody species, which could be added here?
Line 417: The decreased total amino acids contents in leaves and needles of beech and fir observed in the present study cannot be attributed to enhanced growth, exceeding enhanced N acquisition from the soil, since total N contents were not affected by drought exposure in the previous year. Rephrase?
Line 424: … in the presence of the shrub Rubus fruticosus. Is this statement important in the context of this paragraph?
Line 447: Little interaction of interspecific neighbourhood density on growth as well as physiological drought memory is probably due to overweighed complimentary effects rather than competition, as observed in previous studies (Magh, Grün, et al. 2018, Magh, Yang, et al. 2018, Magh et al. 2019, Yang et al. 2022). Maybe it can also be that not growing together from germination onwards may have restricted effects of interspecies neighbourhood.
Line 456: …based on epigenetic processes… Is it an idea to explain the term epigenetics as it is used in this context, in the introduction?
Author Response
Maybe some figures can be put together? E.g. fig 2 and 3, Fig 1 and 4, fig 9, 10 and 11 etc…
Response: Good idea, this would make the manuscript easy to read. We have put Figs1 and 4, Figs 2 and 3, Figs.5, 7 and 8, Figs. 9, 10 and 11 together. In the revised manuscript, there are only 5 figures. Accordingly, the figure legends and text in the manuscript have been updated.
- I wonder in how far the height of the individual seedlings (above ground) influenced their different N contents that were measured. Different statistical methods allow for accounting for the height of the seedlings while comparing drought treated and control groups.
Response: Thank you for this suggestion. We have tried several statistical approaches, but did not receive conclusive results.
- Personally, I would have liked to have seen p-values for the different statistical tests in the text (when mentioning that this or that is/was significantly different from another), but I understand it may make the text more difficult to read.
Response: Yes, you are right. This would make the text more difficult to read, particularly when more than two groups are compared. However, the extents of p values are provided by asterisks, *, **and *** indicating p<0.05, 0.01 and 0.001, respectively.
Some detailed remarks:
Line 82: trigged, maybe it should be triggered?
Response: trigged is replaced by triggered. Please see line 76.
Line 101: The mean lengths of beech and fir seedlings at planting were 21 cm and 31 cm for shoot, 14 cm and 24 cm for root, respectively. How did you measure the “length” of the root? Also, give standard deviations.
Response: The length of the root was measured from the rhizome joint to the tip of the root. Seedlings with similar length and growth were roughly selected before planting. In case a few seedlings with very long roots, the extra roots were cut to minimise hydraulic variations. The standard deviations are given in the manuscript. Please see line 101.
Line 147: FW – DW)/FW; maybe explain what the abbreviations FW and DW stand for (fresh weight, dry weight I suppose)?
Response: This information has been added, please see line 148-149.
Line 188: Data shown are means + S.D. Aren’t the figures normal box plots?
Response: Here and others throughout the manuscript have been corrected to “Data shown are means ± SD”. They are normal box plots generated from SigmaPlot.
Line 202: Significant differences between the number of beech neighbours are shown by different capital letters. I do not see this in the figure?
Response: The legend has been corrected. Please see figure 2 in the revised version.
Line 208: It was also observed, when data were separated according to the number of neighbouring firs, but was not significantly affected by this number (Figs. 2, S2). Maybe rephrase?
Response: Has been rephrased to “Increased biomass of beech was also observed, when data were separated according to the number of neighbouring firs (Figs. 2a, S4).” Please see line 207-208.
Line 212: …a decrease in biomass at 5 neighbouring beech seedlings (Figs. 1, 3, S3). This is not so clear to me when I look at the figures.
Response: Here the text is misleading. The decrease is only shown in Fig. 2b of the revised manuscript. This has been corrected into “A similar effect of drought hardening on biomass accumulation in the subsequent year was not observed for silver fir, but rather a decrease in biomass at 5 neighbouring beech seedlings (Figs. 2b, S5).”. Please see line 208-210.
Line 229: Is there no N in the stems? (idem for C)
Response: N and C contents in stems were not determined.
Line 277: Total amino acid N of European beech and silver fir seedling… Maybe the N should be deleted? Or it should be added in the axis title of the figure and in the main text? Also in line 291
Response: The axis has been amended to “Total amino acid N” and “Soluble protein N”. Please see figure 5 in line 251 in the revised manuscript.
Line 298: In silver fir, structural N in current year needles was reduced, but not significantly affected in previous year needles and roots by drought hardening in the previous year. Highest structural N was recorded in previous year needles; lower but similar contents were found in current year needles and roots (Fig. 11). Maybe rephrase both sentences to make the results more clear?
Response: Has been rephrased to “In silver fir, drought hardening resulted in significantly reduced structural N in current year needles, whereas structural N in previous year needles and roots were not impacted. Structural N content was significantly higher in previous year needles compared to current year needles and roots (Fig. 5c)”. Please see line 265-269.
Line 341: This result is consistent with the present result showing that drought altered carbon contents, observed directly after mild water deprivation and rewetting as mechanism of drought hardening (Yang et al. 2022), were largely reversed in the subsequent year (Fig. 4). Does fig 4 show this statement?
Response: This is a mistake and should refer to Fig. 3a. The mistake has been corrected, please see line 297-298.
Line 344: Moreover, in contrast to the generally decreased root biomass observed in pure beech cultures upon drought (Davidson et al. 1992, Fotelli et al. 2001, Löf 345 et al. 2005, Meier and Leuschner 2008, Rose et al. 2009, Tomasella et al. 2019), the facilitation of fir on beech observed in the previous year (Yang et al. 2022) was also observed in the subsequent year of cultivation (Figs.1, 2, S1 ). “The facilitation of fir on beech “ is not so clear to me.
Response: The facilitation is demonstrated in water relations, C and N contents of leaves. The sentence has been amended to “The facilitation of fir on beech foliar water relations, C and N status observed in the previous year [24] was also observed in the subsequent year of cultivation (Figs.1-3, S1)”. Please see line 299-300.
Line 366:.. phenotypic and morphological effects… Are these two not the same?
Response: The word of “phenotypic” has been deleted. Please see line 316.
Line 378: …observed stimulated post-drought growth… There is also quite some literature on post-drought “compensation growth” in woody species, which could be added here?
Response: Besides the paper of Arend et al. 2016 already cited in the manuscript, two more reference of Bose et al 2021 and Ovenden et al. 2021 have been added, please see line 331.
Line 417: The decreased total amino acids contents in leaves and needles of beech and fir observed in the present study cannot be attributed to enhanced growth, exceeding enhanced N acquisition from the soil, since total N contents were not affected by drought exposure in the previous year. Rephrase?
Response: Has been rephrased to “The decreased total amino acids contents in leaves and needles of beech and fir observed in the present study cannot be attributed to enhanced growth, since total N contents were not affected by previous year drought.” Please see line 363-365.
Line 424: … in the presence of the shrub Rubus fruticosus. Is this statement important in the context of this paragraph?
Response: The words of “in the presence of the shrub Rubus fruticosus” were removed. Please see line 369.
Line 447: Little interaction of interspecific neighbourhood density on growth as well as physiological drought memory is probably due to overweighed complimentary effects rather than competition, as observed in previous studies (Magh, Grün, et al. 2018, Magh, Yang, et al. 2018, Magh et al. 2019, Yang et al. 2022). Maybe it can also be that not growing together from germination onwards may have restricted effects of interspecies neighbourhood.
Response: Previous publications of Magh, Grün, et al. 2018, Magh, Yang, et al. 2018, Magh et al. 2019 studied beech and fir mixtures in the field, where they are growing all the time together, but still no clear interaction of interspecific neighbourhood density was recorded. Therefore, we make this speculation.
Line 456: …based on epigenetic processes… Is it an idea to explain the term epigenetics as it is used in this context, in the introduction?
Response: This statement in line 456 has been deleted. However, this is a very good idea to give this term in the Introduction. Therefore, we added the sentence “As long living organisms, trees may particularly use epigenetics to facilitate phenotypic and physiological modifications in response to environmental changes (Amaral et al. 2020).” Please see line 82-84.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear Author
I have read the revised manuscript (forest-1934123). This manuscript I reviewed the initial draft already. Titled: Memory effects of water deprivation in European beech (Fagus sylvatica L.) and white fir (Abies alba Mill.) seedlings grown in mixed cultivation for publication of forest MDPI. This is the second submission made by the author. The author addressed all the questions and suggestions that I raised the issue in the review of the original manuscript. I satisfy the author’s revisions throughout the paper. The author well-addresses all the questions and quarries in this manuscript. Especially the author improved the introduction and discussion section very well inflow. Now, this manuscript improved the flow of writing, which was comparatively shallow in the original version but in this revised copy author addressed all the quarries and suggestions very well. Before accepting this manuscript if there is anything needed to be revised by the author, especially English grammar, or spell check, I request this manuscript is currently in “Minor Revision” and the author may correct any further grammatical errors (if any) the author may improve in this stage. Thank you.
Author Response
Dear Ms. Lv,
Thanks so much for your quick processing and the fast review of the reviewer. In the present version, we have tried our best to minimise the overlapped contents from previous publication, particularly the Material and Methods part. Moreover, we corrected a mistake in line 210-211. We also checked the cited reference and we confirm that all references are relevant to the contents of the manuscript. For more detailed changes, please see the revised manuscript with Track Changes model.
Best wishes,
Fengli Yang
