Effects of Spring Warming and Drought Events on the Autumn Growth of Larix kaempferi Seedlings

Round 1

Reviewer 1 Report

Your manuscript reports effectively a nice experiment.  I have no major concerns.  The Introduction and Discussion could possibly indicate more the relevance to other species, which might make your paper of interest to more readers.  Also the overlap in the error bars on the figures seem too great for significances reported.  These need to be checked.  However, a minor revision should be adequate to address my points.  I list my commentss below by section and line number. 

 

Abstract: This section looks good. The only issue I have is that it could be helpful on lines 5 and 6 to somehow indicate that there is a combined elevated temperature and drought treatment since that is indicated later in the Abstract..

 

Introduction:  This section is good but could perhaps put the study in a broader context than the species being studied.  The results should be relevant to other species.

 

Line 26.  It seems inconsistent to indicate natural variability due to anthropogenic influences.  Do you mean “and” instead of “due to”?

 

Line 30.  “species” might be better here than “ecosystems”.  

 

Line 54.  “autumn growth response” might be confusing as it could imply growth during the autumn but I assume you mean overall growth as measured in the autumn at the end of the growing season.    

 

Line 72.  I would change “alters” to “is altered”.     

 

Materials and Methods: In this section there needs to be an indication of what other plants if any occurred in the plots.  I assume that there were no other plants, but some weedy species were likely to have started.  Were the plots weeded?  This could probably go best in the second paragraph.

 

Lines 81-82.  “between June and August” might be clearer if reworded as “during June through August” as I assume you mean during that three month period.

 

Lines 89-90.  I am confused here as it seems to indicate there are 20 plots per treatment but I think you mean 20 plots in total (5 replicates of each of the 4 treatments). 

 

Line 106.  I assume the October 30 reading was at the end of the growing season, but it might be good to indicate this specifically here.

 

Line 112.  Was this equation use only for the final sampling or for each month?    

 

Results:  This section is good overall, and all the Figures and Tables are all useful.

 

Figure 3.  I am confused about the significant differences as indicated by the letters.  With so much overlap in the standard error bars I do not understand why there are significant differences.  This needs to be checked.  Are the error bars actually the standard deviation?

 

Figure 4.  I assume these are the values for individual seedling but they could be plot totals.  This needs to be clearly indicated.

 

Discussion and Conclusions:  More about implications for other species could make the work of relevance to more readers.

 

Line 176.  This is a good point, but “durable” does not seem like the best word.  The warming treatment was simply not imposed for nearly as long of a period.      

 

Line 183.  The treatment did not last 5 months.  Maybe you could say something like 5 months after treatment.      

 

Lines 220-222.  I am not sure that extrapolation from stands to young seedlings is useful.  I do think that reduced competition is a good possibility for explaining your results, but it would be useful to give some indication of the likelihood of competition among the seedlings in the plots.  Given that seedlings are 12.5 cm apart and 40-50 cm tall at the end of the study it seems that competition among seedlings was likely.  Do you have any information on lateral spread roots or foliage and whether the crowns were in contact?

    

Line 240.  I think that damage” should be changed to “damaged seedlings”.  I am also not sure that they are so much damaged as simply reduced in growth.      

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

 

The authors of “Effects of Spring Warming and Drought Events on the Autumn Growth of Larix kaempferi Seedlings” report the results of an experiment in which Larix Kampferi seedings were grown under field conditions with exposure to a fully factorial set of warming and water restriction treatments. They found that individual stressors (heating, drought) consistent with a business-as-usual climate change scenario led to reductions in biomass, but their interaction eliminated this effect. In contrast, the combined impact of the two stressors increased mortality, while individual stressors did not.

 

This appears to be a well-designed experiment. Generally, the paper is clearly written and transparent, but see a few line-by-line questions and suggestions below. My main critique is that the authors interpret the results of their well-designed and straightforward factorial experiment in some confusing ways. These are discussed below, but, essentially, it is important to keep in mind that the effect of one treatment (e.g., warming) should be considered in isolation (the warming-only treatment) and insofar as it contributes to interactions (e.g., the combined warming and drought treatment). The authors could be more clear in contrasting the effects of individual stressors against their synergistic impact and could discriminate between the growth-related responses (height, diameter, biomass) and mortality, which are two fundamentally different phenomena in their study.

 

32-33: These are the response variables measured in the study, but they aren’t all equally important in and of themselves. Instead, what the authors seem to be interested in is performance (growth and mortality). Height, root collar diameter, and biomass are all proxies of growth, but don’t need to mentioned in the first paragraph of the paper.

 

91: Is any information about provenance of the seedlings available? Are they assumed to be relatively homogenous genetically?

 

92-93: Why was warming instituted for just two weeks and not consistently across the study period?

 

100-103: This sentence should come before the one beginning “During the manipulation period…” since it explains how environmental conditions were monitored.

 

Table 1: Why is biomass not included here?

 

Table 1: Would it be possible to include a table showing the effect sizes (regression coefficients) for these models as well? It would be helpful especially to have regression output tables for height, RCD, biomass, and mortality – the main response variables. There aren’t too many figures/tables in the current draft, so adding 4 regression output tables in would not hurt.

 

148-158 and Fig. 5: Is this cumulative mortality or mortality per month? Meaning: did 2% (8%-6%) of trees in the combined treatment die between Sept and Oct or did 8% of trees in the combined treatment die between Sept and Oct?

 

160-176: This interpretation does not fully agree with the authors’ results. Yes, drought treatment increased mortality – but it seems like it does so in a way that is enhanced by warming. This is an important insight from the factorial experiment, which the authors contradict in lines 169-176. No, warming did not increase mortality *by itself.* But it appears to enhance the negative impact of drought on survival.

 

187-283: I appreciate the authors’ consideration of the potential positive effects of stress on performance and the extent to which heat stress can prime plants to be more tolerant of other stressors. Other references of interest might include:

 

Epron (1997) Effects of drought on photosynthesis and on the thermotolerance of photosystem II in seedlings of cedar [Cedrus atlantica and C. libani]. Journal of Experimental Botany (48)

 

Ladjal et al. (2000) Effects of drought preconditioning on thermotolerance of photosystem II and susceptibility of photosynthesis to heat stress in cedar seedlings. Tree Physiology (20)

 

Kozlowski et al. (2002) Acclimation and Adaptive Responses of Woody Plants to Environmental Stresses. The Botanical Review (68)

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript “Water-1753395” entitled “Effects of Spring Warming and Drought Events on the Autumn Growth of Larix kaempferi Seedlings” by Kim et al. focuses on an open-field experiment study, which was conducted to evaluate the growth and mortality of Larix kaempferi seedlings in response to spring warming and drought treatments and to determine whether seedlings could regain their growth capability once the treatments were discontinued.

 

It is clear that the authors have done a lot of work, but the manuscript as current written falls considerably short of its promise. The paper suffers from a lack of enough background, rationale, and take-home messages for the readers to understand the novelty of the study. Results are insufficient for publication and need to be revisited for completeness and accuracy. In addition, the findings of the study should be appropriately discussed in the context of previous literature.

 

Thank you for your consideration.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The text has been corrected according to my suggestions. Responses to comments are satisfactory. I recommend the manuscript be published in the Water journal.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dear authors,

I enjoyed reading your manuscript and wish you the best with your future work.

Comments for author File: Comments.docx

Author Response

Responses to Reviewer 1

 

[General comments]

 

Decreased density is unlikely to affect the enhanced growth of seedlings. Authors should provide more evidence to discuss competition as being relevant for the detected growth changes. In fact, 1-year old seedlings are more to be affected by the status of mineral nutrients and soil moisture than by competition and therefore density fluctuation. I strongly advise authors to provide results on competition level among seedlings and on the effect competition has on their growth or mortality. Authors have data and measurements that allow calculating at least distance independent competition indices (size symmetric or asymmetric). In my opinion, the mean growing space of each seedling (170-180 cm2) is large enough, not to be affected by direct competition.

In light-demanding tree species, shading can have important effects on growth and mortality in early life stages. Authors should provide information on how the shading by warming panels (infrared heaters) affected the growth and mortality of seedlings.

▶Response:

Thank you very much for your suggestion. We thought of adding the competition indices in the discussion section, however, we do not have all data to calculate those indices in the current study. Actually we planted 53 1-year-old seedlings per 1 m² and the space between seedlings was approximately 12.5 cm. There were enough spaces for canopy development in the beginning of the study, but we found that the canopy space became decreasing rapidly as seedlings grew. Therefore, we speculated that competition might be one of the potential causes for the current study results. The distance independent competition indices you mentioned must be an important future research topic. We added this point to the conclusion section as follows; “To accurately predict stress response dynamics under climate change, further studies should be conducted on various stress levels, molecular based protection mechanisms, such as the antioxidant systems, and distance independent competition indices.” (Line 241-244).

We also thank you for pointing out the shading effect. Shading is a critical environmental variable determining light resources for seedlings. However, shading by the infrared heater was controlled by establishing the dummy of the infrared heater in the control plots. Therefore, we think that the study results focusing on warming and drought treatments were not affected by the shading effect.

 

[Specific comments]

 

[Comment 1] L26:

please, be more specific on what kind of trends in plants (to predict trends...)

▶Response:

Thank you for your meaningful comment. We materialized the kind of trends in plants as follows; “Therefore, it is essential to predict seedling height, root collar diameter, biomass, and mortality rate under climate change conditions.” (Line 32-33).

 

[Comment 2] L79:

Please, specify how were seedlings planted on the plot, or include a photo documenting the arrangement of seedlings on the plots.
▶Response:

Thank you very much for your comment. We added a figure of seedling arrangement and spacing. Please refer to the new ‘Figure 1. Seedling arrangement and spacing per plot’ marked in red. “Eighty 1-year-old L. kaempferi seedlings were planted in each plot (Figure 1).” (Line 90-91).

 

Figure 1. Seedling arrangement and spacing per plot.

 

[Comment 3] L94:

please, specify if measurements were done on all or on the selected number of seedlings
▶Response:

Thank you for your comment. Actually, measurements were done on all seedlings and accordingly we revised the sentence as follows; “We measured all seedling’s height and root collar diameter on April 30, June 1, and October 30, 2020” (Line 105-106).

 

[Comment 4] L100:

please specify how was the mortality rate calculated
▶Response:

Thank you for your comment. We added the calculation of the mortality rate as follows; “Mortality rate (%) = ” (eq 1).

 

[Comment 5] Line 103:

In fact, air temperature, soil moisture content and soil temperature are independent variables and should be treated as so.
▶Response:

Thank you for your comment. We revised the sentence as follows; “The effects of artificial warming and drought and their interaction on each dependent variable (air temperature, soil temperature, soil moisture content, seedling height, root collar diameter, biomass accumulation, and mortality) were analyzed using two-way ANOVA.” (Line 114-116).

 

[Comment 6] Fig. 1:

please exclude (May 2020) from the axis title. It is enough to provide this information in the figure caption.
▶Response:

Thank you for your comment. We excluded the (May 2020) as follows;

 

 

[Comment 7] Fig. 3,4:

Remove (Nov 2020) from the y, x-axis title and include this information in the figure caption. In Fig. 4 Omit dots in abbreviations of calendar months on the x-axis
▶Response:

We removed the (Nov 2020) and included this information in the figure caption. And we omitted dots in abbreviations of the calendar month on the x-axis in Fig. 5 as follows;

 

Figure 4. Biomass accumulation of Larix kaempferi seedlings as influenced by warming and drought treatments in November 2020.

 

As Figure 1 was newly added, we revised the figure numbers.

 

[Comment 8] L137:

Please include „The relative mortality rate...“
▶Response:

‘Relative mortality’ is defined as the observed number of deaths divided by the expected number of deaths (Elie et al., 2011; Borch-Johnsen et al., 1985). We do not have an expected mortality rate, so it is difficult to obtain a relative mortality rate. If you mean the mortality rate in comparison to the control or the change in the relative mortality rate over time, we think the observed mortality rate is sufficient to represent the study results.

 

 

Reference

 

Borch-Johnsen, K., Andersen, P. K., & Deckert, T. (1985). The effect of proteinuria on relative mortality in type 1 (insulin-dependent) diabetes mellitus. Diabetologia, 28(8), 590-596.

Dai, A. (2013). Increasing drought under global warming in observations and models. Nature climate change, 3(1), 52-58.

Elie, C., De Rycke, Y., Jais, J. P., & Landais, P. (2011). Appraising relative and excess mortality in population-based studies of chronic diseases such as end-stage renal disease. Clinical Epidemiology, 3, 157.

Author Response File: Author Response.docx

Reviewer 2 Report

The MS entitled “Effects of Spring Warming and Drought Events on the Autumn Growth of Larix kaempferi Seedlings” is interesting and reports an open field effect of warming and drought on the growth of Larix seedlings. This could provide a valuable tool for studying underlying mechanisms for abiotic stress signaling. However, the main limitation is the presentation by the authors that makes gets a little focused, especially in the discussion section. The molecular mechanisms-based mechanistic explanation should be discussed instead of superficially covering the subjects.

A concluding line highlighting the outcome of the present study should be added at the end of the abstract.

The introduction is not balanced but should be improved further by adding the latest and most relevant references.

Please mention some important case studies related to the test plant.

There should be a mechanistic explanation in the discussion part.

Conclusion- some future prospectus related to the present study should be given.

 

Author Response

Responses to Reviewer 2

 

[General comments]

 

The MS entitled “Effects of Spring Warming and Drought Events on the Autumn Growth of Larix kaempferi Seedlings” is interesting and reports an open field effect of warming and drought on the growth of Larix seedlings. This could provide a valuable tool for studying underlying mechanisms for abiotic stress signaling. However, the main limitation is the presentation by the authors that makes gets a little focused, especially in the discussion section. The molecular mechanisms-based mechanistic explanation should be discussed instead of superficially covering the subjects.

▶Response:

Thank you very much for your detailed comments and suggestions on this manuscript. We carefully revised the manuscript following your comments. The responses to each of your comments can be found below, and the revisions to the manuscript are presented in red font.

 

[Specific comments]

 

[Comment 1]

A concluding line highlighting the outcome of the present study should be added at the end of the abstract.

▶Response:

Thank you for your comment and suggestion. Please refer to the end of the abstract as follows; “Our study demonstrates that despite the high mortality and the decreased growth under high temperature and drought stress during the treatment period, seedlings showed high levels of resilience compared to seedlings under a single stress.” (Line 15-18).

 

[Comment 2]

The introduction is not balanced but should be improved further by adding the latest and most relevant references.

▶Response:

Thank you very much for your comment. The latest and most relevant references were added to the introduction sections. Please refer to the revised manuscript as follows; “Climate change has generally accelerated the hydrological processes, resulting in more intense and frequent drought events (Mukherjee et al., 2018). Apart from natural climatic variability, the last several decades have experienced severe and protracted droughts due to anthropogenic influences (Dai, 2013).” (Line 24-27).

 

[Comment 3]

Please mention some important case studies related to the test plant.

▶Response:

We33 added some important case studies related to Larix kaempferi as follows; “Noh et al. (2021) showed that extreme climate events such as heatwaves, drought, and heavy rainfall did not affect the survival rate, biomass, shoot-to-root ratio, and seedling quality index of L. kaempferi. In addition, Kwon et al. (2020) showed that the mortality rate of L.kaempferi increased under the increased temperature and decreased precipitation, and the seedling quality index was low in the increased precipitation treated group.” (Line 63-68).

 

[Comment 4]

There should be a mechanistic explanation in the discussion part.

▶Response:

We added a mechanistic explanation as follows; “If cells are exposed to stresses that disrupt homeostasis, the unfolded protein response, endoplasmic reticulum-associated degradation, and the heat shock response will all become active to aid recovery (Wiegant et al., 2013). It has been shown that activating the heat shock response results in increased amounts of heat shock proteins, which alleviate a load of aberrant proteins by enabling them to refold their original shape (Morimoto et al., 1997). Cells also demonstrate higher concentrations of components from the ubiquitin-proteasome system after being exposed to stress, and as a result, they have a greater ability to destroy proteins that cannot be refolded as a result (Amm et al., 2014). The modulation of heat shock protein levels, overexpression of ubiquitin-proteasome system components, may be stated to be a reliable molecular basis for the positive effects of hormesis (Wiegant et al., 2013).” (Line 193-202).

 

[Comment 5]

Conclusion- some future prospectus related to the present study should be given.

▶Response:

Thank you for your comment. We added some future prospectus as follows; “Therefore, even if 1-year-old L. kaempferi seedlings experience spring warming and drought events at a similar level to this study in the future, it is expected or speculated that the damage will sufficiently recover when they are allowed to experience normal temperature and precipitation in summer and autumn. To accurately predict stress response dynamics under climate change, further studies should be conducted on various stress levels, molecular based protection mechanisms, such as the antioxidant systems, and distance independent competition indices.” (Line 237-244).

 

 

Reference

 

Amm, I., Sommer, T., & Wolf, D. H. (2014). Protein quality control and elimination of protein waste: the role of the ubiquitin–proteasome system. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(1), 182-196.

Dai, A. (2013). Increasing drought under global warming in observations and models. Nature climate change, 3(1), 52-58.

Morimoto, R. I., Kline, M. P., Bimston, D. N., & Cotto, J. J. (1997). The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays in biochemistry, 32, 17-29.

Mukherjee, S., Mishra, A., & Trenberth, K. E. (2018). Climate change and drought: a perspective on drought indices. Current Climate Change Reports, 4(2), 145-163.

Wiegant, F. A. C., Poot, S. D., Boers-Trilles, V. E., & Schreij, A. M. A. (2013). Hormesis and cellular quality control: a possible explanation for the molecular mechanisms that underlie the benefits of mild stress. Dose-Response, 11(3), dose-response.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The MS has been improved. Therefore I recommend accept.