Morphological, Physiological, and Transcriptional Responses to Drought Stress in Sensitive and Tolerant Elm (Ulmus pumila L.) Varieties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, 

Thanks much for providing such a research work,  I went over the manuscript,  found some errors and areas needs additional enhancements as follows: 

1. Abstract

Issues Identified:

Overly dense and lacks clear segmentation for objectives, methods, and results.

No explicit emphasis on the broader implications of findings.

Redundant mention of some physiological traits like soluble sugars and proteins, which could be condensed.

Recommendations:

Simplify and segment the abstract to improve readability.

Focus on the key findings and their applications without excessive technical detail.

2. Introduction

Issues Identified:

Repetitive discussion on the impact of drought stress, particularly in paragraphs 2 and 3.

Abrupt transition from general drought stress context to the specific objectives of the study.

Insufficient citations for some claims, such as the molecular mechanisms of drought resistance in Ulmus pumila.

Recommendations:

Eliminate redundancy and improve flow by integrating related concepts.

Add citations for unreferenced claims to strengthen the scientific rigor.

3. Materials and Methods

Issues Identified:

Lack of justification for experimental design choices, such as selecting 20 cultivars and specific physiological parameters.

Environmental conditions like light intensity and humidity during the experiment are not detailed.

Table 1 lacks clarity in describing why specific locations were chosen for sampling.

Recommendations:

Include a rationale for the selection of cultivars and physiological measurements.

Provide additional details about experimental conditions for reproducibility.

4. Results

Issues Identified:

Figures and tables lack comprehensive captions, making them hard to interpret independently.

Statistical significance is mentioned but without sufficient quantifiable backing (e.g., p-values, confidence intervals).

Overlap between textual and tabular/graphical data leads to redundancy.

Recommendations:

Enhance figure and table captions with detailed explanations of their purpose.

Present statistical results with proper quantifiable details.

Avoid repeating data in multiple formats (text and visuals).

5. Discussion

Issues Identified:

Abrupt transitions between topics, such as from RWC to ABA signaling, disrupt the logical flow.

Overgeneralization in some interpretations, such as describing ULP-19 and ULP-12 as "stronger" without detailed quantification.

Insufficient connection between transcriptome data and physiological observations.

Recommendations:

Reorganize the discussion to group related themes more cohesively.

Provide specific data points to support generalized claims.

Expand on the implications of transcriptome findings in the context of drought resistance.

6. Conclusions

Issues Identified:

Overly brief and fails to highlight the broader applications or implications of the findings.

Redundant repetition of results already discussed in detail.

Recommendations:

Expand the conclusions to include potential practical applications of the findings.

Avoid reiterating results; instead, focus on the implications for future research and agricultural practices.

7. References

Issues Identified:

Inconsistent formatting of references, particularly in-text citations ([1, 2] style varies in appearance).

Some sources, such as “[3],” are too vague, lacking adequate detail to verify.

Recommendations:

Ensure all references follow the journal's specified format.

Verify that all in-text citations correspond accurately to the reference list.

8. Figures and Tables

Issues Identified:

Figures like Figure 1 and 2 lack standalone clarity due to insufficient captions.

Statistical markers (e.g., error bars, significance asterisks) are unclear in some charts.

Tables contain excessive numerical data without adequate explanation of its relevance.

Recommendations:

Add detailed captions to figures and tables for better interpretation.

Clarify statistical markers and ensure consistency across visuals.

Consider summarizing numerical data in key points rather than listing exhaustive values.

9. Language and Grammar

Issues Identified:

Occasional grammatical inconsistencies, such as tense shifts (e.g., "Weigh 0.2 g of fresh leaves" switches tense unexpectedly).

Repeated use of jargon-heavy phrases that may be difficult for non-specialist readers.

Recommendations:

Maintain consistent verb tense throughout the manuscript.

Simplify overly technical language to improve accessibility.

Author Response

Responses to Reviewer 1’s comments:

Comments and Suggestions for Authors

Dear Authors, 

Thanks much for providing such a research work, I went over the manuscript, found some errors and areas needs additional enhancements as follows: 

1. Abstract

Issues Identified:

Overly dense and lacks clear segmentation for objectives, methods, and results.

No explicit emphasis on the broader implications of findings.

Redundant mention of some physiological traits like soluble sugars and proteins, which could be condensed.

Recommendations:

Simplify and segment the abstract to improve readability.

Focus on the key findings and their applications without excessive technical detail.

2. Introduction

Issues Identified:

Repetitive discussion on the impact of drought stress, particularly in paragraphs 2 and 3.

Abrupt transition from general drought stress context to the specific objectives of the study.

Insufficient citations for some claims, such as the molecular mechanisms of drought resistance in Ulmus pumila.

Recommendations:

Eliminate redundancy and improve flow by integrating related concepts.

Add citations for unreferenced claims to strengthen the scientific rigor.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. For improving the quality of the manuscript, we have made the following modifications to the manuscript:

For figures, to provide comprehensive environmental information for the provenance of the 20 selected elm cultivars, we have incorporated annual precipitation (AP) and annual mean temperature (AMT) data into Table 1. The original Figure 1 has been removed, and the former Figure 2 is now relabeled as Figure 1 to illustrate physiological indicators. Additionally, a new Figure 2 has been added, showcasing the phenotypic images of elm trees from the control and drought-stressed groups of the drought-tolerant and sensitive cultivars identified by physiological metrics. This figure further highlights the effects of drought stress on the leaves and roots of the four selected varieties. We also eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

For manuscript, in accordance with the reviewers' suggestions, we have thoroughly revised the abstract, introduction, discussion, and conclusion sections. In the abstract, we highlighted the key findings of the study and clarified the underlying mechanisms of drought tolerance in the selected cultivars. The introduction and discussion sections were structured based on the following logic: Drought stress induces water deficiency in plants, leading to stomatal closure in leaves to minimize water loss. However, the stomatal closure inhibits CO₂ fixation and photosynthesis. The stable drought resistance of drought-resistant plants/cultivar is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones. In the conclusion, we summarized the important findings and emphasized the practical significance of this study.

 

3. Materials and Methods

Issues Identified:

Lack of justification for experimental design choices, such as selecting 20 cultivars and specific physiological parameters.

Table 1 lacks clarity in describing why specific locations were chosen for sampling.

Recommendations:

Include a rationale for the selection of cultivars and physiological measurements.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We added the reasons for the selection of cultivars and physiological measurements. As:

“The 20 elm cultivars selected for this experiment were collected from diverse environmental conditions across China. These include severely cold and arid regions characterized by an annual precipitation (AP) of less than 200 mm and an annual mean temperature (AMT) below 12°C, such as ULP18, ULP19, and ULP20; moderately cold and arid regions with AP < 500 mm and AMT < 8°C, including ULP11, ULP12, and ULP13; and relatively warm and slightly arid regions with AP < 800 mm and AMT < 15°C, exemplified by ULP2, ULP3, and ULP10 (Table 1).”

“The experiment was conducted in a natural field habitat homogeneity garden. Solar radiation and humidity data for the experimental area over the past decade were obtained from the meteorological website (https://wheata.cn/). The total solar radiation intensity in the experimental area ranged from 182.15 to 197.24 W/m². During the experiment, the average air humidity ranged from 45% to 80%.”

“The physiological indicators selected in this study were primarily based on the literature concerning drought stress research in Wheat, Solanum rostratum, Oak, and eleven other tree species [21-23].”

 

4. Results

Issues Identified:

Figures and tables lack comprehensive captions, making them hard to interpret independently.

Statistical significance is mentioned but without sufficient quantifiable backing (e.g., p-values, confidence intervals).

Overlap between textual and tabular/graphical data leads to redundancy.

Recommendations:

Enhance figure and table captions with detailed explanations of their purpose.

Present statistical results with proper quantifiable details.

Avoid repeating data in multiple formats (text and visuals).

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions.

We modified the figure and table captions. As:

“Table 1. Sampling information of 20 elm cultivars from areas with varying degrees of drought and cold in China”

“Figure 1. Physiological and phenotypic indicators of 20 elm cultivars under drought stress.”

“Table 2. Drought tolerance evaluation of four elm cultivars by membership functions”

“Figure 2. Phenotypic observations of the aboveground and underground parts of the rep-resentative cultivars, the growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3.”

“Table3 Up- and down-regulation of gene expression encoding key enzymes in drought-resistant processes in the roots and leaves of drought-tolerant ULP19 and drought-sensitive ULP2 cultivars.”

We have added the information of statistical tests in the description of Figure 1. As:

“The differences in phenotypic and physiological indicators between the control group and the drought treatment group were analyzed using one-way ANOVA. The P-values are presented as asterisks above the box plots. **** indicates P-value < 0.001, *** indicates P-value < 0.005, ** indicates P-value < 0.01, * indicates P-value < 0.05, and ns indicates no significant difference.”

We also eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

 

5. Discussion

Issues Identified:

Abrupt transitions between topics, such as from RWC to ABA signaling, disrupt the logical flow.

Overgeneralization in some interpretations, such as describing ULP-19 and ULP-12 as "stronger" without detailed quantification.

Insufficient connection between transcriptome data and physiological observations.

Recommendations:

Reorganize the discussion to group related themes more cohesively.

Provide specific data points to support generalized claims.

Expand on the implications of transcriptome findings in the context of drought resistance.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have rewritten the discussion section, mainly highlighting the logical relationships between each paragraph: summarizing the main results based on phenotypic, physiological and transcriptome sequencing, and distill the key points. Drought-tolerant cultivars possessed more efficient water regulation mechanisms, however their internal water content still decreased relative to the control sample. ULP-19 enhances drought tolerance by effectively regulating stomatal closure via ABA regulation. The stable drought resistance of ULP-19 is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots. Under drought stress, ULP-19 enhances starch degradation to compensate for the reduced soluble carbohydrates resulting from suppressed Calvin-Benson cycle activity in low CO₂ conditions in leaves.

 

6. Conclusions

Issues Identified:

Overly brief and fails to highlight the broader applications or implications of the findings.

Redundant repetition of results already discussed in detail.

Recommendations:

Expand the conclusions to include potential practical applications of the findings.

Avoid reiterating results; instead, focus on the implications for future research and agricultural practices.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We rewrote the conclusion section, as:

“By evaluating phenotypic and growth indicators, two drought-tolerant varieties (ULP-19 and ULP-12) and two sensitive ones (ULP-2 and ULP-3) were determined. Transcriptome sequence analysis shows that the stable drought resistance of drought-resistant variety ULP-19 was associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones (ABA). These insights will facilitate the choice of suitable elm varieties for reforestation projects and contribute to the establishment of sustainable forest ecosystems in arid region. In future studies, we should compare the intensity of photosynthesis and the con-tents of both soluble carbohydrates and starch in different organs of drought-resistant and drought-sensitive elm plants prior to the onset of drought stress.”

 

7. References

Issues Identified:

Inconsistent formatting of references, particularly in-text citations ([1, 2] style varies in appearance).

Some sources, such as “[3],” are too vague, lacking adequate detail to verify.

Recommendations:

Ensure all references follow the journal's specified format.

Verify that all in-text citations correspond accurately to the reference list.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We checked and modified the references.

 

8. Figures and Tables

Issues Identified:

Figures like Figure 1 and 2 lack standalone clarity due to insufficient captions.

Statistical markers (e.g., error bars, significance asterisks) are unclear in some charts.

Tables contain excessive numerical data without adequate explanation of its relevance.

Recommendations:

Add detailed captions to figures and tables for better interpretation.

Clarify statistical markers and ensure consistency across visuals.

Consider summarizing numerical data in key points rather than listing exhaustive values.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We modified the captions, legends of all tables and figures.

We have added the information of statistical tests and details in the legends of Figure 1 and Figure 2. As:

“Figure 1. Physiological and phenotypic indicators of 20 elm cultivars under drought stress. The root collar diameter (a) and plant height (b) index of 20 elm cultivars. The root (c) and leaf relative water content (d) and leaf chlorophyll content (e) of tolerant and sensitive cultivars; the SOD (f), soluble sugar (g) and soluble protein content (h) of tolerant and sensitive cultivars leaves. CK represents the control group, and DS represents the drought treatment group. The differences in phenotypic and physiological indicators between the control group and the drought treat-ment group were analyzed using one-way ANOVA. The P-values are presented as asterisks above the box plots. **** indicates P-value < 0.001, *** indicates P-value < 0.005, ** indicates P-value < 0.01, * indicates P-value < 0.05, and ns indicates no significant difference.”

“Figure 2. Phenotypic observations of the aboveground and underground parts of the representative cultivars, the growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. (a) The above-ground phenotypes of four cultivars under drought stress were marked by red arrows. ULP-19 and ULP-12 plants maintained similar leaf numbers to the control group, with yellowing limited to basal leaves. In contrast, ULP-2 and ULP-3 showed a significant decrease in leaf number and more extensive yellowing from mid to basal portions. (b) Compared to ULP-2 and ULP-3, ULP-19 and ULP-12 exhibit a more extensive lateral and fibrous root system, characterized by longer root lengths, as indicated by the yellow arrows. The box plots of yellow leaf number (c) and root biomass (d) for the four cultivars revealed significant differences among them. One-way ANOVA results, with P-values indicated above the respective box plots, showed that ULP-19 and ULP-12 had significantly fewer yellow leaves and higher root biomass com-pared to ULP-2 and ULP-3.”

 

 

9. Language and Grammar

Issues Identified:

Occasional grammatical inconsistencies, such as tense shifts (e.g., "Weigh 0.2 g of fresh leaves" switches tense unexpectedly).

Repeated use of jargon-heavy phrases that may be difficult for non-specialist readers.

Recommendations:

Maintain consistent verb tense throughout the manuscript.

Simplify overly technical language to improve accessibility.

 

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We modified the grammar and tenses of the entire text have been revised. As:

“Weighed 0.2 g of fresh leaves, added 10 mL of pH 7.8 phosphate buffer, ground the mixture to a homogenate, centrifuged at 4000 rpm for 15 min, and collected the supernatant as the sample extract.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

It is a pleasure for me to have reviewed the manuscript titled “Morphological, physiological, and transcriptional responses to drought stress in sensitive and tolerant elm (Ulmus pumila) cultivars.” The manuscript is interesting and relevant to a wide audience, both in the field and in the laboratory. However, there are sections that require improvement to enhance the quality of the document.

Abstract
The abstract is adequate but would benefit from incorporating quantitative results to support the claims. Additionally, I suggest adding a concluding sentence that highlights the relevance of the study to the field, which would help establish the importance of the research.

Introduction
The authors have provided a comprehensive introduction that clearly contextualizes the study, but there are some formatting errors that need correction:

1. development[10]: The citation should be separated from the word.
2. Arabidopsis thaliana: This scientific name should be italicized.
3. While the introduction is well-structured, analyzing various elements, it could be enriched by expanding on the motivation to study Ulmus pumila, not only for its ecological significance but also its economic potential and role in mitigating climate change.
4. Justify the importance of combining morphological, physiological, and transcriptomic data to achieve a comprehensive understanding of drought tolerance.
5. Highlight more explicitly the current knowledge gaps, especially concerning molecular mechanisms of tolerance in specific cultivars.

Materials and Methods

The methodology includes a significant amount of critical information; however, some points require clarification:

1. It is not specified how uniform conditions were ensured for the seeds prior to planting, which could influence comparisons among cultivars.
2. I suggest revising the wording “are” to “were” since this refers to a past event: The collected seeds of the 20 cultivars “were” planted.
3. The type of soil used in the planting pits is not mentioned, nor are details about fertility or texture analyses.
4. Although the average precipitation and temperature are mentioned, there is no information on how these variables were monitored or controlled during the experiment.
5. The planting of 15 seedlings per pit raises questions about whether spatial distribution affects plant competition.
6. In Table 1, the meaning of "ULP" should be described below the table for immediate clarification.
7. When referring to cultivars, are these truly cultivars or accessions? If they are cultivars, they should have established names rather than generic labels like ULP-1 to ULP-20.
8. 2.2. Growth Measurement: Although tree growth was minimal, using a ruler might introduce measurement errors. A hypsometer would yield more accurate results.
9. 2.3. Sampling: While the information is clear, there is no explanation as to why ULP19 and ULP12 were selected as representatives of drought-tolerant cultivars and ULP2 and ULP3 as sensitive ones. The selection criteria must be clearly stated.
10. Root sampling lacks detail; it is unclear how roots were selected.
11. 2.4. Physiological Traits Measurement:
• This section requires more depth so readers can replicate the experiments.
• Specify whether biological replicates were used. For fresh weight measurements, indicate whether leaves were cleaned prior to weighing.
• Chlorophyll measurements lack information about controls. SOD analyses do not mention how light conditions were controlled.
• 2.4.5. Leaf Soluble Protein Content: This section needs more precision; reagent concentrations are not specified.
• The methodology should detail the equipment used, including brand and country of manufacture, to facilitate replication.
12. 2.6. RNA Extraction:
• As mentioned earlier, the criteria for tree selection for sampling should be detailed.
• Explain how samples were processed before RNA extraction and how RNA integrity was maintained to prevent degradation.
• puri-fied should be corrected to purified.
• Specify the software used for transcriptomic analysis and the parameters applied.
• Ensure all scientific names follow proper formatting rules.
• Software and version details should be explicitly mentioned.
• I recommend moving Figure 1 to the Results section.

Results
The experiment evaluates conditions and identifies sensitive trees, but the parameters used for this classification are not mentioned beyond phenotypic observations. Measured parameters are needed to support these claims.

1. “The growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. For ULP19 and ULP12, there were fewer senescing leaves at the plant bottom, and there are more lateral and fibrous roots in the root system, with longer lengths.” While the description is clear, this section requires objectivity. Interpretation is better suited for the Discussion. Include specific results for each measurement, as quantitative data are essential for establishing comparisons.

Discussion
The authors have provided an interesting discussion, but several weaknesses and issues need to be addressed to improve clarity, scientific rigor, and coherence in the conclusions:

1. Images should be significantly improved.
2. Tables should be reviewed (e.g., Table 3 is labeled as “Table3”). Capitalize appropriately (e.g., “cellulose” should be “Cellulose”).
3. The selection process for sensitive and tolerant cultivars before the experiments is not explained. This is crucial since the results and interpretations rely on this classification.
4. Results on aspects such as height, stem growth, chlorophyll content, and osmotic substance accumulation need clearer definitions. How significant were the reductions in height or root growth? Quantitative values are necessary for objective comparisons.
5. While some related studies are cited, the comparison with them is superficial. Discuss how results differ or align with previous studies regarding the magnitude and observed mechanisms.
6. Unsupported interpretations, such as cultivars having "more comprehensive photosynthetic protection mechanisms" or "better water regulation," need to be substantiated with specific indicators.
7. The discussion generalizes differences between sensitive and tolerant cultivars but does not address potential exceptions or variations within each group. Avoid overgeneralizing; instead, discuss whether observations are consistent across all cultivars in each group.
8. Mention specific genes (e.g., OST1, HXK1, SUS4/5/6), but clarify how their differential expression translates to observed phenotypes. Include concrete data (e.g., gene expression levels) and explicitly connect these observations to phenotypic changes.
9. Limitations of the study, such as variability in experimental conditions, the limited number of cultivars, or the lack of replication in other environmental contexts, are not mentioned.
10. The discussion covers multiple topics (chlorophyll content, osmotic substance accumulation, gene expression, etc.) but lacks a clear narrative thread. Better structuring would guide the reader.
11. Gene data (e.g., CRK29, DPE1, etc.) are presented in isolation and not well-integrated with phenotypic and physiological results. Make the connections more explicit and evidence-based.
12. The conclusion that "tolerant cultivars have more efficient regulatory mechanisms" seems overly generalized based on limited data. Provide more evidence to support this claim.

13.    Conclusions
While the conclusion is somewhat grounded in the results, it lacks integration of the study’s importance, limitations, and future research directions.

References
The manuscript cites limited literature for the study. Expanding the discussion with more references would address this issue.

1. References are inconsistent and should be standardized.
2. Reference 7 does not italicize the scientific name.
3. Reference 37 lacks uniformity.

 

Comments for author File: Comments.pdf

Author Response

Responses to Reviewer 2’s comments:

Dear Editor,

It is a pleasure for me to have reviewed the manuscript titled “Morphological, physiological, and transcriptional responses to drought stress in sensitive and tolerant elm (Ulmus pumila) cultivars.” The manuscript is interesting and relevant to a wide audience, both in the field and in the laboratory. However, there are sections that require improvement to enhance the quality of the document.

Abstract
The abstract is adequate but would benefit from incorporating quantitative results to support the claims. Additionally, I suggest adding a concluding sentence that highlights the relevance of the study to the field, which would help establish the importance of the research.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. For improving the quality of the manuscript, we have made the following modifications to the manuscript:

For figures, to provide comprehensive environmental information for the provenance of the 20 selected elm varieties, we have incorporated annual precipitation (AP) and annual mean temperature (AMT) data into Table 1. The original Figure 1 has been removed, and the former Figure 2 is now relabeled as Figure 1 to illustrate physiological indicators. Additionally, a new Figure 2 has been added, showcasing the phenotypic images of elm trees from the control and drought-stressed groups of the drought-tolerant and sensitive varieties identified by physiological metrics. This figure further highlights the effects of drought stress on the leaves and roots of the four selected varieties. We also eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

For manuscript, in accordance with the reviewers' suggestions, we have thoroughly revised the abstract, introduction, discussion, and conclusion sections. In the abstract, we highlighted the key findings of the study and clarified the underlying mechanisms of drought tolerance in the selected varieties. The introduction and discussion sections were structured based on the following logic: Drought stress induces water deficiency in plants, leading to stomatal closure in leaves to minimize water loss. However, the stomatal closure inhibits CO₂ fixation and photosynthesis. The stable drought resistance of drought-resistant plants/variety is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones. In the conclusion, we summarized the important findings and emphasized the practical significance of this study.

We rewrote the abstract section. As:

“The exploration of genetic resources of drought-resistant trees is highly significant for improving the ecological environment. Elm trees are widely distributed in the cold and arid regions of the Northern Hemisphere and have a strong tolerance for adverse conditions. This study evaluated the drought tolerance of 20 elm varieties (Ulmus pumila L.) collected from areas with varying de-grees of drought and cold. By assessing phenotypic and growth indicators, we found that drought-sensitive varieties ULP-2 and ULP-3 exhibited a significant reduction in height (31 cm and 16 cm) and root collar diameter (0.37 cm and 0.17 cm), whereas drought-tolerant varieties ULP-19 and ULP-12 maintained favorable growth conditions under drought stress. Moreover, ULP-19 and ULP-12 displayed significantly fewer yellow leaves (12 and 19) and higher root biomass (237.8 g and 221.2 g).  Compared with the sensitive varieties, the relative water content in leaves (21.02 % and 30.10 %) and roots (26.13% and 34.53) decreased less in the drought-tolerant varieties. Tran-scriptome sequence analysis shows that the stable drought resistance of drought-resistant varietie ULP-19 is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones (abscisic acid). These results will aid in the selection of appropriate elm varieties for afforestation initiatives and the development of sustainable forest ecosystems in arid region.”

 

Introduction
The authors have provided a comprehensive introduction that clearly contextualizes the study, but there are some formatting errors that need correction:

1. development[10]: The citation should be separated from the word.
2. Arabidopsis thaliana: This scientific name should be italicized.
3. While the introduction is well-structured, analyzing various elements, it could be enriched by expanding on the motivation to study Ulmus pumila, not only for its ecological significance but also its economic potential and role in mitigating climate change.
4. Justify the importance of combining morphological, physiological, and transcriptomic data to achieve a comprehensive understanding of drought tolerance.
5. Highlight more explicitly the current knowledge gaps, especially concerning molecular mechanisms of tolerance in specific varieties.

Responses:

We are very grateful for the reviewers' comments and have rewritten the introduction section. The introduction sections were structured based on the following logic: Drought stress induces water deficiency in plants, leading to stomatal closure in leaves to minimize water loss. However, the stomatal closure inhibits CO₂ fixation and photosynthesis. The stable drought resistance of drought-resistant plants/trees is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones.

“As a naturally occurring herb, it holds significant economic value, particularly in Asian traditional medicine. In traditional medical practices, humans utilize the stem and root of U. pumila to address various health conditions, including edema, mastitis, gastric cancer, and inflammation [15,16]. Consistent with its medicinal applications, numerous bioactive compounds have been isolated from U. pumila extracts [12,16-18].”

 

Materials and Methods

The methodology includes a significant amount of critical information; however, some points require clarification:

1. It is not specified how uniform conditions were ensured for the seeds prior to planting, which could influence comparisons among cultivars.

Responses:

Thanks. We have already modified this item. “Seeds were collected from their respective provenance areas during the mature season of the year preceding sowing. Collection times varied based on local temperature conditions: in regions with an annual average temperature below 8-12 °C, seeds were gathered around June, whereas in regions with an annual average temperature above 15 °C, collection occurred from mid-April to early May. Prior to experimentation, all seeds were verified to be at a consistent stage of maturity: 50 days after flowering (DAF), 14-15 mm in length, and yellow color.”

1. I suggest revising the wording “are” to “were” since this refers to a past event: The collected seeds of the 20 cultivars “were” planted.

Responses:

Thanks. We have already modified this item.

1. The type of soil used in the planting pits is not mentioned, nor are details about fertility or texture analyses.

Responses:

Thanks. We have already modified this item. “The soil mainly consisted of a mixture of sandstone, sand, mudstone, and gravel, with a pH of 6.5.

1. Although the average precipitation and temperature are mentioned, there is no information on how these variables were monitored or controlled during the experiment.

Responses:

Thanks. We have already modified this item. “Throughout the study period, the air temperature (HMP155A, Campbell Scientific Inc., Logan, UT, USA; n=15) and soil temperature at 10 cm depth (S-TMB, HOBO S-LIA, Onset Computer Corporation; n = 25) were measured. Measurements indicated that during the experimental month, air temperature fluctuated between 5 and 28 °C, while soil temperature varied between 6 and 25 °C.”

1. The planting of 15 seedlings per pit raises questions about whether spatial distribution affects plant competition.

Responses:

Since the stress treatment experiment was conducted within a single growing season, the experimental design considered the growth range of the plants during this period to minimize competition among them. During the collection of root samples, no significant root-to-root contact was observed.

1. In Table 1, the meaning of "ULP" should be described below the table for immediate clarification.

Responses:

Thanks. We have already modified this item. “ULP= Ulmus pumila”

1. When referring to cultivars, are these truly cultivars or accessions? If they are cultivars, they should have established names rather than generic labels like ULP-1 to ULP-20.

Responses:

Thank you for the reviewers' suggestions. The materials we used were collected from the natural habitats in the wild and did not have clear variety names. We expected to screen and name the variety names through this experiment. Therefore, we have changed it to "varieties" in accordance with the suggestions of multiple reviewers.

1. 2.2. Growth Measurement: Although tree growth was minimal, using a ruler might introduce measurement errors. A hypsometer would yield more accurate results.

Responses:

Thanks. We have already modified this item. In this experiment, both the ruler and the hypsometer were used to measure the height of the trees. We have revised the description accordingly. As:

“the meter ruler and hypsometer were used to directly measure the total height of the seedlings. Total height is the height of the elm seedlings from its stump to its tiptop.”

1. 2.3. Sampling: While the information is clear, there is no explanation as to why ULP19 and ULP12 were selected as representatives of drought-tolerant cultivars and ULP2 and ULP3 as sensitive ones. The selection criteria must be clearly stated.

Responses:

“2.3. Sampling

Thanks. We have already modified this item. We classify the drought resistant ratio greater than 0.5 as drought-tolerant varieties, and those less than 0.5 as drought-sensitive varieties.”

1. “Root sampling lacks detail; it is unclear how roots were selected.

Responses:

Thanks. We have already modified this item. “When measuring the physiological indicators of roots, select roots with a diameter 1-2 mm, thoroughly wash them, and then proceed with the measurements.”

1. 2.4. Physiological Traits Measurement:
• This section requires more depth so readers can replicate the experiments.
• Specify whether biological replicates were used. For fresh weight measurements, indicate whether leaves were cleaned prior to weighing

Responses:

Thanks. We have already modified this item. “Fresh leaves were cleaned to remove soil and other contaminants, and were cut and crushed. Before measuring the physiological indicators, the leaves were thoroughly washed and air-dried. For leaf samples, three biological replicates were established.”

• Chlorophyll measurements lack information about controls. SOD analyses do not mention how light conditions were controlled.

Responses:

Thanks. We have already modified this item. “Extract solution (50 mL; Vethanol: Vacetone = 1:1) was added to 0.2 g of control and drought stress treated leaves. As controls, tubes without sample homogenates were analyzed in parallel.”

• 2.4.5. Leaf Soluble Protein Content: This section needs more precision; reagent concentrations are not specified.
• The methodology should detail the equipment used, including brand and country of manufacture, to facilitate replication.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. Due to the word limit imposed by the journal, specific details, parameters, and reagent concentrations for physiological experiments such as SOD activity and protein content were referenced from the corresponding literature. In this manuscript, only key information is provided, while detailed experimental procedures are omitted.

“The physiological indicators selected in this study were primarily based on the literature concerning drought stress research in Wheat, Solanum rostratum, Oak, and eleven other tree species [21-23].”

1. 2.6. RNA Extraction:
• As mentioned earlier, the criteria for tree selection for sampling should be detailed.

Responses:

Thanks. We have already modified this item. “Trees with growth characteristics, including plant height (208 cm/210 cm for ULP-19 CK/DS, 250 cm/219 cm for ULP-2 CK/DS), rd (2.65 cm/2.66 cm for ULP-19 CK/DS, 3.02 cm/2.66 cm for ULP-2 CK/DS), and number of yellow leaves (12 for ULP-19 DS, 34 for ULP-2 DS), that were closest to the mean values were selected as representative specimens for each varieties. For sampling, green and healthy leaves, along with fine roots measuring 1-2 mm in diameter, were chosen. The selected leaves and roots were meticulously cleaned, rinsed with water, and subsequently dried.”

• Explain how samples were processed before RNA extraction and how RNA integrity was maintained to prevent degradation.

Responses:

Thanks. We have already modified this item. “Due to the word limit imposed by the journal, standard RNA quality assessment and evaluation methods, which are widely documented elsewhere, have been omitted from this manuscript. The detailed RNA extraction and RNA integrity protocols referred to this website (https://rna.cd-genomics.com/resource/rna-extraction-guide.html).

• puri-fied should be corrected to purified.

Responses:

Thanks. We have already modified this item.     

• Specify the software used for transcriptomic analysis and the parameters applied.
• Ensure all scientific names follow proper formatting rules.
• Software and version details should be explicitly mentioned.

Responses:

Thanks. We have already modified this item. “Gene expression levels were estimated by RSEM v1.3.3 [27] for all samples. The clean reads were aligned to the de novo assembled transcriptome.”

“The GO enrichment analysis for the differentially expressed genes (DEGs) in elm samples were per-formed by the topGO v1.0 package of R. The DEGs of elm unigene IDs were transferred to the Arabidopsis TAIR locus IDs during the MapMan analysis. The software KOBAS v3.0 were used to test the statistical enrichment of differential expression genes in KEGG pathways in elm [29].”

• I recommend moving Figure 1 to the Results section.

Responses:

Thanks. The original Figure 1 has been removed, and the former Figure 2 is now relabeled as Figure 1 to illustrate physiological indicators.

Results
The experiment evaluates conditions and identifies sensitive trees, but the parameters used for this classification are not mentioned beyond phenotypic observations. Measured parameters are needed to support these claims.

1. “The growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. For ULP19 and ULP12, there were fewer senescing leaves at the plant bottom, and there are more lateral and fibrous roots in the root system, with longer lengths.” While the description is clear, this section requires objectivity. Interpretation is better suited for the Discussion. Include specific results for each measurement, as quantitative data are essential for establishing comparisons.

Responses:

Thanks. We have already modified this item. “Drought resistance of 20 varieties index by root collar diameter (Rd) and plant height are shown in Figure 1a-b. Drought significantly reduced root collar diameter and plant height in most varieties, the decrease rates were 5.77%- 16.06% and 2.24%- 13.73%. However, the patterns of reduction of five varieties were not significant: ULP-11, ULP-12, ULP-13, ULP-14 and ULP-19. The plant height of three varieties (ULP-12, ULP-13 and ULP-19) were not significantly different between the treatments.

Phenotypic observations of the aboveground and underground parts of the representative varieties showed significant differences under drought or well-watered conditions (Figure 2 a, b). ULP-19 and ULP-12 plants maintained similar leaf numbers to the control group, with yellowing limited to basal leaves. In contrast, ULP-2 and ULP-3 showed a significant decrease in leaf number and more extensive yellowing from mid to basal portions (Figure 2 a). Compared to ULP-2 and ULP-3, ULP-19 and ULP-12 exhibit a more extensive lateral and fibrous root system, characterized by longer root lengths, as indicated by the yellow arrows (Figure 2 b). Besides, based on quantitative phenotypic indicators, the mean number of yellow leaves for ULP-19 and ULP-12 was less than 20, while that for ULP-2 and ULP-3 was more than 30 (Figure 2 c). There was also a significant difference in root biomass between the two groups of plants. The mean root biomass for ULP-19 and ULP-12 was greater than 220g, while that for ULP-3 was 160g and for ULP-2 was 135g (Figure 2 d).”

“To evaluate the drought resistance of the four elm varieties, six physiological indexes under continuous drought stress and rehydration conditions were analyzed by member-ship functions (Table 2). The comprehensive evaluation value of the four elm varieties was calculated as mean value of membership functions of six physiological indexes. Higher comprehensive evaluation values were positively correlated with stronger drought-resistance ability. The results showed that the drought resistant ratio of ULP-19, ULP-12, ULP-2 and ULP-3 were 1.000, 0.596, 0.060 and 0.229. The order of drought re-sistance of the four elm varieties as follows: ULP19 > ULP12 > ULP3 > ULP2. We classify the drought resistant ratio greater than 0.5 as drought-tolerant varieties, and those less than 0.5 as drought-sensitive varieties.”

Discussion
The authors have provided an interesting discussion, but several weaknesses and issues need to be addressed to improve clarity, scientific rigor, and coherence in the conclusions:

1. Images should be significantly improved.
2. Tables should be reviewed (e.g., Table 3 is labeled as “Table3”). Capitalize appropriately (e.g., “cellulose” should be “Cellulose”).

Responses:

Thanks. We have made the following modifications to the charts in the entire text.

For figures, to provide comprehensive environmental information for the provenance of the 20 selected elm varieties, we have incorporated annual precipitation (AP) and annual mean temperature (AMT) data into Table 1. The original Figure 1 has been removed, and the former Figure 2 is now relabeled as Figure 1 to illustrate physiological indicators. Additionally, a new Figure 2 has been added, showcasing the phenotypic images of elm trees from the control and drought-stressed groups of the drought-tolerant and sensitive varieties identified by physiological metrics. This figure further highlights the effects of drought stress on the leaves and roots of the four selected varieties. We also eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

1. The selection process for sensitive and tolerant cultivars before the experiments is not explained. This is crucial since the results and interpretations rely on this classification.
2. Results on aspects such as height, stem growth, chlorophyll content, and osmotic substance accumulation need clearer definitions. How significant were the reductions in height or root growth? Quantitative values are necessary for objective comparisons.

Responses:

“In this study, we investigated the effects of drought stress on the growth characteris-tics of elm varieties. We classify varieties with a drought resistance ratio greater than 0.5 as drought-tolerant and those less than 0.5 as drought-sensitive. ULP19 and ULP12 were se-lected as representative drought-tolerant elm varieties, while ULP2 and ULP3 represent the drought-sensitive varieties. The results showed that drought-sensitive varieties ULP-2 and ULP-3 exhibited a significant reduction in height (31 cm for ULP-2; 16 cm for ULP-3) and root collar diameter (0.37 cm for ULP-2; 0.17 cm for ULP-3), whereas drought-tolerant varieties ULP-19 and ULP-12 maintained favorable growth conditions under drought stress (Figure 1 a, b). Moreover, ULP-19 and ULP-12 displayed significantly fewer yellow leaves (12 for ULP-19; 19 for ULP-12) and higher root biomass (237.8 g for ULP-19; 221.2 g for ULP-12) (Figure 2 c, d). Through a comprehensive analysis of key pathways and gene expression patterns in both above-ground and underground tissues of the drought-tolerant varieties ULP-19, we found that drought conditions prompted this varieties to reorganize its carbon allocation and metabolism.”

 

1. While some related studies are cited, the comparison with them is superficial. Discuss how results differ or align with previous studies regarding the magnitude and observed mechanisms.
2. Unsupported interpretations, such as cultivars having "more comprehensive photosynthetic protection mechanisms" or "better water regulation," need to be substantiated with specific indicators.
3. The discussion generalizes differences between sensitive and tolerant cultivars but does not address potential exceptions or variations within each group. Avoid overgeneralizing; instead, discuss whether observations are consistent across all cultivars in each group.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have rewritten the discussion section, mainly highlighting the logical relationships between each paragraph: summarizing the main results based on phenotypic, physiological and transcriptome sequencing, and distill the key points. Drought-tolerant varieties possessed more efficient water regulation mechanisms, however their internal water content still decreased relative to the control sample. ULP-19 enhances drought tolerance by effectively regulating stomatal closure via ABA regulation. The stable drought resistance of ULP-19 is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots. Under drought stress, ULP-19 enhances starch degradation to compensate for the reduced soluble carbohydrates resulting from suppressed Calvin-Benson cycle activity in low CO₂ conditions in leaves.

1. Mention specific genes (e.g., OST1, HXK1, SUS4/5/6), but clarify how their differential expression translates to observed phenotypes. Include concrete data (e.g., gene expression levels) and explicitly connect these observations to phenotypic changes.

Responses:

Thanks. We added the expression levels of the key genes in different samples behind them. “Drought stress induces water deficiency in plants, leading to stomatal closure in leaves to minimize water loss. Transcriptome sequence analysis reveals that ULP-19 enhances drought tolerance by effectively regulating stomatal closure. OST1 (Clus-ter-34330.17145, the mean values of three repeated FPKM, ULP-19 leaves = 69.76, ULP-2 leaves = 14.24, ULP-19 roots = 0.03, ULP-2 roots = 0.15), a calcium-independent ABA-activated protein kinase, plays a central role in stomatal regulation by responding to ABA signals and triggering downstream reactions that influence stomatal opening and closing [31]. ALDH3H1 (Cluster-34330.26768, 2.33, 0, 1.07, 0.31), an aldehyde dehydrogenase, may be involved in processing ABA metabolic intermediates, thus regulating ABA signal transduction [32]. CRK29 (Cluster-34330.25113, 10.39, 2.62, 1.40, 0), on the contrary, is a cysteine-rich receptor-like protein kinase that can regulate its activity by phosphorylating other proteins, thereby influencing ABA signal transduction and amplification [33]. The upregulation of these genes in ULP-19 leaves suggests that this drought-tolerant cultivar reduces water loss through enhanced ABA regulation and stomatal closure.”

1. Limitations of the study, such as variability in experimental conditions, the limited number of cultivars, or the lack of replication in other environmental contexts, are not mentioned.

Responses:

Thanks. We added the limitations, as: “Although this study provides valuable references for the screening of drought-tolerant elm varieties and elucidating their molecular basis through phenotypic, physiological, and transcriptome sequencing, it also highlights several limitations, such as variability in experimental conditions, the limited number of varieties, or the lack of replication in other environmental contexts. In future studies, we should compare the intensity of photosynthesis and the contents of both soluble carbohydrates and starch in different organs of drought-resistant and drought-sensitive elm varieties prior to the onset of drought stress.”

1. The discussion covers multiple topics (chlorophyll content, osmotic substance accumulation, gene expression, etc.) but lacks a clear narrative thread. Better structuring would guide the reader.
2. Gene data (e.g., CRK29, DPE1, etc.) are presented in isolation and not well-integrated with phenotypic and physiological results. Make the connections more explicit and evidence-based.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have rewritten the discussion section, mainly highlighting the logical relationships between each paragraph: summarizing the main results based on phenotypic, physiological and transcriptome sequencing, and distill the key points. Drought-tolerant varieties possessed more efficient water regulation mechanisms, however their internal water content still decreased relative to the control sample. ULP-19 enhances drought tolerance by effectively regulating stomatal closure via ABA regulation. The stable drought resistance of ULP-19 is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots. Under drought stress, ULP-19 enhances starch degradation to compensate for the reduced soluble carbohydrates resulting from suppressed Calvin-Benson cycle activity in low CO₂ conditions in leaves.

 

1. The conclusion that "tolerant cultivars have more efficient regulatory mechanisms" seems overly generalized based on limited data. Provide more evidence to support this claim.

13. Conclusions
    While the conclusion is somewhat grounded in the results, it lacks integration of the study’s importance, limitations, and future research directions.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We rewrote the conclusion section, as:

“By evaluating phenotypic and growth indicators, two drought-tolerant varieties (ULP-19 and ULP-12) and two sensitive ones (ULP-2 and ULP-3) were determined. Transcriptome sequence analysis shows that the stable drought resistance of drought-resistant variety ULP-19 was associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones (ABA). These insights will facilitate the choice of suitable elm varieties for reforestation projects and contribute to the establishment of sustainable forest ecosystems in arid region. In future studies, we should compare the intensity of photosynthesis and the contents of both soluble carbohydrates and starch in different organs of drought-resistant and drought-sensitive elm plants prior to the onset of drought stress.”

References
The manuscript cites limited literature for the study. Expanding the discussion with more references would address this issue.

1. References are inconsistent and should be standardized.
2. Reference 7 does not italicize the scientific name.
3. Reference 37 lacks uniformity.

 Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We checked and modified the references.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The present manuscript by Zheng et al. is devoted to studying of the causes of drought resistance in elm (Ulmus pumila) cultivars. The results of the study and the value of the research are beyond doubt.

-         However, the conclusions of the study should be clarified. In the conclusions the researchers should divide the observed changes into those that relate directly to the plant strategy of drought resistance and regulation of these processes. Calcium ions often serve as primary sensors of various stresses. Further development of the signal leads to a change in the hormonal status of plants. In the case of drought, abscisic acid takes part in this process, changing the expression profile of plants, initiating the cascade of protective reactions, such as increased activity of antioxidant enzymes, synthesis of lignin and various osmolytes and other reactions. In this sense it seems better to rephrase the last issue to “Transcriptome sequence analysis shows that the stable drought resistance of drought-resistant varieties is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones (abscisic acid).” The authors, of course, can state the conclusion differently, but “plant hormones (abscisic acid), calcium ions, transcriptional regulation in leaves, carbohydrate metabolism and lignin synthesis in roots” should not be listed as just “associated” with the robust drought tolerance of drought-tolerant cultivars.

 -         Significant attention in the Discussion section is given to carbohydrate metabolism, and this is very correct, since drought should lead to a change in the intensity of the processes of degradation and synthesis of carbohydrates, since it is necessary to increase the content of carbohydrates in the leaves in order to reduce water loss. But the primary problem under drought conditions is that the stomata close. This process is, in fact, controlled by abscisic acid and limits the supply of CO2 necessary for the process of photosynthesis. An interesting question that the authors might consider in the Discussion is where drought-tolerant elm plants get their carbohydrates after stomata close. Perhaps they transport them by degrading starch stored in roots. In this sense, I agree with the statement that “Drought conditions prompt plants to reorganize their carbon allocation and metabolism”, but I disagree with “causing more carbon to be transferred from leaves to roots and other key organs and altering the composition of carbohydrates, such as increasing starch and fructose content and decreasing sucrose content [37]”, because leaves are not able to produce carbohydrates after stomata closing. Concerning the reference [37]”, Cuellar-Ortiz et al. (2008) did not conduct any studies on the distribution of carbohydrates between leaves and roots. They studied the processes associated with the outflow of carbohydrates to the seeds. In the present manuscript it is also said “Meanwhile, the expression of genes involved in starch degradation, such as BAM2 and CT-BMY, indicates the initiation of energy storage, which is crucial for maintaining metabolic activity when photosynthesis is impaired [39]”. Please, indicate, if BAM2 and CT-BMY expression was increased. If it is so, this is additional an evidence to what was written above: starch degradation is enhanced, which helps compensate for the lack of soluble carbohydrates due to the suppressed functioning of the Calvin-Benson cycle in conditions of low CO2 in leaves.

 -         Figure 1 c-j. The photos of the aboveground and underground phenotypes of ULP19, ULP12, ULP3 and ULP2 cultivars after drought stress treatment are shown before it appeared that ULP19, ULP12 cultivars are drought tolerant and ULP3 and ULP2 cultivars are drought sensitive. Thus, these data are not clear to a reader and, moreover, they are not described in the text. These Figures should be given after the Figure 2, they should be supplemented with data from photographs of control conditions and the features of the organization of the leaf crown and root system of control plants, drought-resistant and drought-sensitive plants should be described in the text. At that, in the Discussion section it is said that “The results indicated that the drought-sensitive cultivars ULP-2 and ULP 3 had a significant reduction in height and stem growth, while the drought-tolerant cultivars ULP-19 and ULP-12 could maintain good growth conditions under drought. Additionally, the drought-sensitive cultivars showed leaf curling and yellowing and a significant inhibition of root growth under drought stress. In contrast, the drought-tolerant cultivars were also affected to some extent, but the phenotypic changes were relatively minor, demonstrating stronger adaptability and resistance.”  First, please, don’t forget to put references to the Figures and Tables of the present study after the statements. It should be the references to the data of Fig. 1c,d,g,h, Fig. 1 e,f,i,j and Figure 2a, b. The second is that I don’t see that “the drought-sensitive cultivars showed leaf curling and yellowing and a significant inhibition of root growth under drought stress.” Please, add the correspondent photos.

-         Besides, in page 7 it is not indicated in any way that ULP-19 and ULP-12 were chosen as were chosen drought-tolerant cultivars, and ULP-2 and ULP 3 were chosen as drought-sensitive ones. It becomes clear more later. Please, correct.

-         Figure 2. “CK” and “DS” abbreviations should be indicated in the Figure legend.

 -         “Phenotypic observations of the aboveground and underground parts of the representative cultivars showed significant differences under drought or well-watered conditions (Figure 1 c, d). The growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. For ULP19 and ULP12, there were fewer senescing leaves at the plant bottom and there are more lateral and fibrous roots in the root system, with longer lengths.” Concerning the growth pattern, I can see that it was better for ULP19 and ULP12 from the data of the Figures 2a, b. Fewer senescing leaves at the plant bottom and more lateral and fibrous roots in the root system should be clearer indicated in the photographs. Perhaps it would be worthwhile to indicate them with arrows or provide more illustrative photographs.

 -         Figure 3 legend. “Gene expression pattern and functional transition over the time course.” What was the time? Please, indicate. It is shown the Gene expression pattern and functional transition in ULP19 and ULP2 leaves and roots. We can find it only written by small letters in the left corner of the Figure. Please, add this information to the legend.

 -         Page 10.  “This study compared the RWC changes of four elm cultivars under drought conditions and found that the drought-tolerant cultivars ULP-19 and ULP-12 had a smaller decrease in RWC under drought stress than the sensitive cultivars ULP-2 and ULP-3.” Please, add the reference to Figure 2e.

 -         Page 10. “Moreover, this study discovered that drought stress generally led to a decrease in chlorophyll content in all four elm cultivars. However, the chlorophyll content of the drought-tolerant cultivars was relatively higher, further suggesting that their photosynthesis protection mechanisms are more complete and can better maintain photosynthesis efficiency under drought conditions.”  It is a very common misconception to equate the chlorophyll content with the intensity of photosynthetic processes. In fact, often, the reduction of the light-collecting antenna under stress is also one of the many protective mechanisms of degradation of the photosynthetic apparatus, primarily photosystem 2. In the case of this study, most likely, the decrease in chlorophyll and, in general, the yellowing of the leaves is associated with the destruction of chloroplasts and even whole leaves due to starvation. Photoprotective mechanisms apparently have nothing left to save. I would recommend the Authors of the present study to compare the intensity of photosynthesis and the content of carbohydrates, both soluble and starch, in different organs of the drought-resistant and drought-sensitive elm plants before the onset of drought in their future studies. It seems that drought-resistant cultivars were already prepared to survive unfavorable conditions before they occurred.

-         Table 3. “The key drought-resistant processes and genes in the root of drought-tolerant cultivar ULP19.” It seems, it should be “Up- and down-regulation of the expression of the genes encoding the enzymes participating the key drought-resistant processes in the roots and leaves of drought-tolerant cultivar ULP19 and drought-sensitive cultivar ULP2”. GlcA - please give the complete name.

-         Figure 4a. The Figure is unreadable. Improve the quality and indicate which main genes belong to the specified clusters. Are these genes in Table 3? I can't assess this, since I can't see the letters in the Figure. If these are other genes, perhaps it would be worth making an additional Table in the Supplementary. Moreover, the Figure legend indicates “genes”, while there are the clusters in the Figure 4a.

-         The legend to the Figure 4b. “The key genes of carbohydrate metabolic pathway related with drought resistance in ULP 19 roots.” These are not the genes, the enzymes encoding by the genes are in the Figure, since the metabolic processes are depicted.  It should be noted that they are marked in red. Am I correct in understanding that they all are upregulated? All abbreviations used in the figure must be explained in the figure caption.

-         “2.3. Sampling”: “A total of three disease- and pest free leaves were select from each elm tree.” It seems, it should be “selected”.

Author Response

Responses to Reviewer 3’s comments:

The present manuscript by Zheng et al. is devoted to studying of the causes of drought resistance in elm (Ulmus pumila) cultivars. The results of the study and the value of the research are beyond doubt.

-         However, the conclusions of the study should be clarified. In the conclusions the researchers should divide the observed changes into those that relate directly to the plant strategy of drought resistance and regulation of these processes. Calcium ions often serve as primary sensors of various stresses. Further development of the signal leads to a change in the hormonal status of plants. In the case of drought, abscisic acid takes part in this process, changing the expression profile of plants, initiating the cascade of protective reactions, such as increased activity of antioxidant enzymes, synthesis of lignin and various osmolytes and other reactions. In this sense it seems better to rephrase the last issue to “Transcriptome sequence analysis shows that the stable drought resistance of drought-resistant varieties is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones (abscisic acid).” The authors, of course, can state the conclusion differently, but “plant hormones (abscisic acid), calcium ions, transcriptional regulation in leaves, carbohydrate metabolism and lignin synthesis in roots” should not be listed as just “associated” with the robust drought tolerance of drought-tolerant cultivars.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. For improving the quality of the manuscript, we have made the following modifications to the manuscript:

For figures, to provide comprehensive environmental information for the provenance of the 20 selected elm varieties, we have incorporated annual precipitation (AP) and annual mean temperature (AMT) data into Table 1. The original Figure 1 has been removed, and the former Figure 2 is now relabeled as Figure 1 to illustrate physiological indicators. Additionally, a new Figure 2 has been added, showcasing the phenotypic images of elm trees from the control and drought-stressed groups of the drought-tolerant and sensitive varieties identified by physiological metrics. This figure further highlights the effects of drought stress on the leaves and roots of the four selected varieties. We also eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

For manuscript, in accordance with the reviewers' suggestions, we have thoroughly revised the abstract, introduction, discussion, and conclusion sections. In the abstract, we highlighted the key findings of the study and clarified the underlying mechanisms of drought tolerance in the selected varieties. The introduction and discussion sections were structured based on the following logic: Drought stress induces water deficiency in plants, leading to stomatal closure in leaves to minimize water loss. However, the stomatal closure inhibits CO₂ fixation and photosynthesis. The stable drought resistance of drought-resistant plants/variety is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots, which is determined by increased regulation of transcription in the leaves of protective pathways controlled by calcium ions and phytohormones. In the conclusion, we summarized the important findings and emphasized the practical significance of this study.

 

 -         Significant attention in the Discussion section is given to carbohydrate metabolism, and this is very correct, since drought should lead to a change in the intensity of the processes of degradation and synthesis of carbohydrates, since it is necessary to increase the content of carbohydrates in the leaves in order to reduce water loss. But the primary problem under drought conditions is that the stomata close. This process is, in fact, controlled by abscisic acid and limits the supply of CO2 necessary for the process of photosynthesis. An interesting question that the authors might consider in the Discussion is where drought-tolerant elm plants get their carbohydrates after stomata close. Perhaps they transport them by degrading starch stored in roots. In this sense, I agree with the statement that “Drought conditions prompt plants to reorganize their carbon allocation and metabolism”, but I disagree with “causing more carbon to be transferred from leaves to roots and other key organs and altering the composition of carbohydrates, such as increasing starch and fructose content and decreasing sucrose content [37]”, because leaves are not able to produce carbohydrates after stomata closing. Concerning the reference [37]”, Cuellar-Ortiz et al. (2008) did not conduct any studies on the distribution of carbohydrates between leaves and roots. They studied the processes associated with the outflow of carbohydrates to the seeds. In the present manuscript it is also said “Meanwhile, the expression of genes involved in starch degradation, such as BAM2 and CT-BMY, indicates the initiation of energy storage, which is crucial for maintaining metabolic activity when photosynthesis is impaired [39]”. Please, indicate, if BAM2 and CT-BMY expression was increased. If it is so, this is additional an evidence to what was written above: starch degradation is enhanced, which helps compensate for the lack of soluble carbohydrates due to the suppressed functioning of the Calvin-Benson cycle in conditions of low CO2 in leaves.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have rewritten the discussion section, mainly highlighting the logical relationships between each paragraph: summarizing the main results based on phenotypic, physiological and transcriptome sequencing, and distill the key points. Drought-tolerant varieties possessed more efficient water regulation mechanisms, however their internal water content still decreased relative to the control sample. ULP-19 enhances drought tolerance by effectively regulating stomatal closure via ABA regulation. The stable drought resistance of ULP-19 is associated with changes in the processes of carbohydrate metabolism and lignin synthesis in the roots. Under drought stress, ULP-19 enhances starch degradation to compensate for the reduced soluble carbohydrates resulting from suppressed Calvin-Benson cycle activity in low CO₂ conditions in leaves.

 

 -         Figure 1 c-j. The photos of the aboveground and underground phenotypes of ULP19, ULP12, ULP3 and ULP2 cultivars after drought stress treatment are shown before it appeared that ULP19, ULP12 cultivars are drought tolerant and ULP3 and ULP2 cultivars are drought sensitive. Thus, these data are not clear to a reader and, moreover, they are not described in the text. These Figures should be given after the Figure 2, they should be supplemented with data from photographs of control conditions and the features of the organization of the leaf crown and root system of control plants, drought-resistant and drought-sensitive plants should be described in the text. At that, in the Discussion section it is said that “The results indicated that the drought-sensitive cultivars ULP-2 and ULP 3 had a significant reduction in height and stem growth, while the drought-tolerant cultivars ULP-19 and ULP-12 could maintain good growth conditions under drought. Additionally, the drought-sensitive cultivars showed leaf curling and yellowing and a significant inhibition of root growth under drought stress. In contrast, the drought-tolerant cultivars were also affected to some extent, but the phenotypic changes were relatively minor, demonstrating stronger adaptability and resistance.”  First, please, don’t forget to put references to the Figures and Tables of the present study after the statements. It should be the references to the data of Fig. 1c,d,g,h, Fig. 1 e,f,i,j and Figure 2a, b. The second is that I don’t see that “the drought-sensitive cultivars showed leaf curling and yellowing and a significant inhibition of root growth under drought stress.” Please, add the correspondent photos.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We revised the figures by removing Figure 1 and adding a modified version of Figure 2, which highlights the differences in aboveground and underground phenotypes between drought-sensitive and drought-tolerant varieties. Additionally, we introduced new box plots (Figure 2 c, d) showing the number of yellow leaves and root biomass for each variety after drought stress, providing quantitative evidence of the phenotypic differences between these varieties. As:

“In this study, 30 individuals from each of the four strains (ULP-19, ULP-12, ULP-2, and ULP-3) were selected post-drought stress treatment for analysis. The analysis included quantifying the proportion of yellow leaves and measuring root biomass. The proportion of yellow leaves was determined by calculating the ratio of leaves with more than 50% yellowing area to the total number of leaves. Root biomass was assessed by collecting all roots from the underground parts of the plants. After being subjected to blanching at 105℃ for 1 hour followed by drying at 65℃ until a constant weight was achieved, the biomass was subsequently weighed using an analytical balance with a precision of 0.01%.”

“Figure 2. Phenotypic observations of the aboveground and underground parts of the rep-resentative varieties, the growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. (a) The above-ground phenotypes of four varieties under drought stress were marked by red arrows. ULP-19 and ULP-12 plants maintained similar leaf numbers to the control group, with yellowing limited to basal leaves. In con-trast, ULP-2 and ULP-3 showed a significant decrease in leaf number and more extensive yellowing from mid to basal portions. (b) Compared to ULP-2 and ULP-3, ULP-19 and ULP-12 exhibit a more extensive lateral and fibrous root system, characterized by longer root lengths, as indicated by the yellow arrows. The box plots of yellow leaf number (c) and root biomass (d) for the four varieties revealed significant differences among them. One-way ANOVA results, with P-values indicated above the respective box plots, showed that ULP-19 and ULP-12 had significantly fewer yellow leaves and higher root biomass compared to ULP-2 and ULP-3.”

“In this study, we investigated the effects of drought stress on the growth characteris-tics of elm varieties. We classify varieties with a drought resistance ratio greater than 0.5 as drought-tolerant and those less than 0.5 as drought-sensitive. ULP19 and ULP12 were selected as representative drought-tolerant elm varieties, while ULP2 and ULP3 represent the drought-sensitive varieties. The results showed that drought-sensitive varieties ULP-2 and ULP-3 exhibited a significant reduction in height (31 cm and 16 cm) and root collar diameter (0.37 cm and 0.17 cm), whereas drought-tolerant varieties ULP-19 and ULP-12 maintained favorable growth conditions under drought stress (Figure 1 a, b; Figure 2 a). Moreover, ULP-19 and ULP-12 displayed significantly fewer yellow leaves (12 and 19) and higher root biomass (237.8 g and 221.2 g) (Figure 2 c, d). Through a comprehensive analysis of key pathways and gene expression patterns in both above-ground and under-ground tissues of the drought-tolerant variety ULP-19, we found that drought conditions prompted this variety to reorganize its carbon allocation and metabolism.”

 

-         Figure 2. “CK” and “DS” abbreviations should be indicated in the Figure legend.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. The full names of these two abbreviations have been added to the legend. As:

“CK represents the control group, and DS represents the drought treatment group.”

 

 -         “Phenotypic observations of the aboveground and underground parts of the representative cultivars showed significant differences under drought or well-watered conditions (Figure 1 c, d). The growth pattern of ULP19 and ULP12 under drought conditions was better than that of ULP2 and ULP3. For ULP19 and ULP12, there were fewer senescing leaves at the plant bottom and there are more lateral and fibrous roots in the root system, with longer lengths.” Concerning the growth pattern, I can see that it was better for ULP19 and ULP12 from the data of the Figures 2a, b. Fewer senescing leaves at the plant bottom and more lateral and fibrous roots in the root system should be clearer indicated in the photographs. Perhaps it would be worthwhile to indicate them with arrows or provide more illustrative photographs.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We revised the figures by removing Figure 1 and adding a modified version of Figure 2, which highlights the differences in aboveground and underground phenotypes between drought-sensitive and drought-tolerant varieties. Additionally, we introduced new box plots (Figure 2 c, d) showing the number of yellow leaves and root biomass for each varietie after drought stress, providing quantitative evidence of the phenotypic differences between these varietys. The above-ground phenotypes of both the control and drought-treated groups, as well as the underground phenotypes of the drought-treated groups, for the four strains have been highlighted with red or yellow arrows.

 

 -         Figure 3 legend. “Gene expression pattern and functional transition over the time course.” What was the time? Please, indicate. It is shown the Gene expression pattern and functional transition in ULP19 and ULP2 leaves and roots. We can find it only written by small letters in the left corner of the Figure. Please, add this information to the legend.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have added the information of statistical tests and details in the legend of Figure 3. As:

“Figure 3. Gene expression pattern and functional transition between ULP-19 and ULP-2 leaves and roots. (a) Expression patterns of 12,445 differentially expressed genes (DEGs) genes in eight DEGs clusters. The number of genes in each cluster is shown on the right. (b) Gene Ontology (GO) enriched in eight DEGs clusters. The important processes that are associated with drought resistance were displayed. Genes numbers of the biological pro-cesses are marked in pink or red, with darker shades indicating stronger links to drought tolerance in leaves (C7 and C8) or roots (C4 and C5) of ULP-19.”

 

 -         Page 10.  “This study compared the RWC changes of four elm cultivars under drought conditions and found that the drought-tolerant cultivars ULP-19 and ULP-12 had a smaller decrease in RWC under drought stress than the sensitive cultivars ULP-2 and ULP-3.” Please, add the reference to Figure 2e.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We add the reference to Figure 1 c, d. As:

“This study compared the RWC changes in four elm varieties under drought conditions and found that drought-tolerant varieties ULP-19 and ULP-12 exhibited a smaller de-crease in RWC under drought stress compared to the drought-sensitive varieties ULP-2 and ULP-3 (Figure 1 c, d).”

 

 -         Page 10. “Moreover, this study discovered that drought stress generally led to a decrease in chlorophyll content in all four elm cultivars. However, the chlorophyll content of the drought-tolerant cultivars was relatively higher, further suggesting that their photosynthesis protection mechanisms are more complete and can better maintain photosynthesis efficiency under drought conditions.”  It is a very common misconception to equate the chlorophyll content with the intensity of photosynthetic processes. In fact, often, the reduction of the light-collecting antenna under stress is also one of the many protective mechanisms of degradation of the photosynthetic apparatus, primarily photosystem 2. In the case of this study, most likely, the decrease in chlorophyll and, in general, the yellowing of the leaves is associated with the destruction of chloroplasts and even whole leaves due to starvation. Photoprotective mechanisms apparently have nothing left to save. I would recommend the Authors of the present study to compare the intensity of photosynthesis and the content of carbohydrates, both soluble and starch, in different organs of the drought-resistant and drought-sensitive elm plants before the onset of drought in their future studies. It seems that drought-resistant cultivars were already prepared to survive unfavorable conditions before they occurred.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. To ensure logical coherence, we removed the section discussing chlorophyll and incorporated the future research directions suggested by the reviewers at the end of the discussion. As:

“Although this study provides valuable references for the screening of drought-tolerant elm varieties and elucidating their molecular basis through phenotypic, physiological, and transcriptome sequencing, it also highlights several limitations, such as variability in experimental conditions, the limited number of cultivars, or the lack of replication in other environmental contexts. In future studies, we should compare the intensity of photosynthesis and the contents of both soluble carbohydrates and starch in different organs of drought-resistant and drought-sensitive elm varieties prior to the onset of drought stress.”

 

-         Table 3. “The key drought-resistant processes and genes in the root of drought-tolerant cultivar ULP19.” It seems, it should be “Up- and down-regulation of the expression of the genes encoding the enzymes participating the key drought-resistant processes in the roots and leaves of drought-tolerant cultivar ULP19 and drought-sensitive cultivar ULP2”. GlcA - please give the complete name.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have modified the title of Table 3 and added the full name of GlcA. As:

“Table3 Up- and down-regulation of gene expression encoding key enzymes in drought-resistant processes in the roots and leaves of drought-tolerant ULP19 and drought-sensitive ULP2 varieties.”

 

-         Figure 4a. The Figure is unreadable. Improve the quality and indicate which main genes belong to the specified clusters. Are these genes in Table 3? I can't assess this, since I can't see the letters in the Figure. If these are other genes, perhaps it would be worth making an additional Table in the Supplementary. Moreover, the Figure legend indicates “genes”, while there are the clusters in the Figure 4a.

-         The legend to the Figure 4b. “The key genes of carbohydrate metabolic pathway related with drought resistance in ULP 19 roots.” These are not the genes, the enzymes encoding by the genes are in the Figure, since the metabolic processes are depicted.  It should be noted that they are marked in red. Am I correct in understanding that they all are upregulated? All abbreviations used in the figure must be explained in the figure caption.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We eliminated the original Figure 4 due to its redundancy with the textual content and Table 3.

 

-         “2.3. Sampling”: “A total of three disease- and pest free leaves were select from each elm tree.” It seems, it should be “selected”.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We modified this, as:

“A total of three disease- and pest free leaves were selected from each elm tree.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, 

Thanks much for improving the quality of the provided manuscript addressing positively with my comments. 

Author Response

We sincerely thank the reviewer for constructive criticisms and valuable suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have submitted a revised manuscript that incorporates the suggestions I previously provided. I encourage them to prioritize careful control of experimental conditions in future studies. While this can be challenging in fieldwork, greater attention to experimental design will significantly enhance the consistency of results. Nevertheless, due to the notable improvements in the quality, clarity, and presentation of the information in the manuscript, it is now ready for publication.

Author Response

We sincerely thank the reviewer for constructive criticisms and valuable suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors!

Thank you for your careful analysis of the comments sent. A lot of corrections have been made to improve the manuscript, and new Figures have appeared, which make the material clearer. Unfortunately, now there is some mess with Figures. Some Figures are duplicated, including new ones, and the legends to the Figures 3 and 4 have been lost. Besides, according to the rules of the MDPI, the Materials and Methods section should be after the Discussion section. The authors will still have to move it to the end of the text. Perhaps it makes sense to do this right away and modify Figure 1, extracting the photos 1a and 1b and making it as Figure 5. In any case, the mess with the Figures should be eliminated.

 

The sentence “When measuring the physiological indicators of roots, select roots with a diameter 1-2 mm, thoroughly wash them, and then proceed with the measurements” seems unclear from the point of view of English grammar. Did you mean “When measuring the physiological parameters of roots, roots with a diameter of 1–2 mm are selected, washed thoroughly, and then measurements are taken”?

Author Response

Thank you again for your valuable suggestions. The version you reviewed includes the revisions with track changes enabled. We recommend that you examine the latest revised manuscript, where several updates have been made to the figures. Specifically, the original Figure 1 has been removed and replaced with a new bar chart presenting phenotypic and physiological data. Figure 2 now illustrates the phenotypes of both aboveground and underground parts along with the newly added quantitative bar chart. Figure 3 displays the differentially expressed clusters from the transcriptome analysis and their associated functions. The previous Figure 4 has been eliminated. Additionally, we have revised the sentence regarding the measurement of root physiological parameters according to your recommendations. Thank you for your continued support.