Physiological and Flavonoid Metabolic Responses of Black Locust Leaves to Drought Stress in the Loess Plateau of China

Comments 1: Please check L32, replace "upregulated,." by "upregulated."

Response 1: We have revised this typo.

Revised text (L34):

“upregulated”

Comments 2: Please check L38, replace "reecological protection strategiessponse" by "re-ecological protection strategies response"

Response 2: We have revised this expression.

Revised text (L40):

“re-ecological protection strategies response”

Comments 3:L60 please put in italic  "Black locust" and replace "(black LocustL.)" by "(Robinia pseudoacacia L.)"

Response 3: nk you for pointing this out. We have modified this expression, and adjusted the layout of the introduction part according to reviewer 2's opinions, and the modified position has been adjusted to 97 lines.

Revised text (L94):

“Black locust (Robinia pseudoacacia L.),”

Comments 4:Between L106 and L107 check the correct scientif name of plant specie Robinia pseudoacacia and put in italic letters

Response 4: :We have revised the expression to make it more correct and normative.”black locust”.

Comments 5:We have revised this sentence to make it more clear.

Response 5: Thank you for pointing this out. We agree with this comment.We have revised the expression to make it more correct and normative.

Comments 6: L112 separate the words "locustforests"

Response 6: We have modified the expression.

Revised text (L119):

“black locust forests”

Comments 7:L126 separate the words "locustwas"

Response 7: We have modified the expression.

Revised text (L134):

“locust was”

Comments 8: L127 separate the words "locustin"

Response 8: We have modified the expression.

Revised text (L102):

“locust in”

Comments 9: L138 separate the words "locustforest"

Response 9: We have revised the expression.

Revised text (L147):

“black locust forests”

Comments 10: L139 separate the words "locustin"

Response 10: We have revised the expression.

“locust in”

Comments 11: L141 separate the words "custto"

Response 11: We have revised the expression.

Revised text (L150):

“locust to”

Comments 12: L141 put in italic letter the scientific name of plant specie

Response 12: We have revised the expression to make it more correct and normative.

Revised text (L142-144):

“The intensity of drought stress to black locust was controlled by implementing 40% and 80% drainage exclusion (representing moderate and extreme drought, respectively) (see methods for details.”

Comments 13: L182 please replace double dot by unique dot. Also in L195

Response 13: Thank you for pointing this out.We have removed the superfluous punctuation.

Comments 14: L234 replace "nthrone" by "anthrone"

Response 14: We have revised the expression.

Revised text (L255):

“anthrone”

Comments 15: L245 replace "locustwere" by "Locust were"

Response 15: We have revised the expression.

Revised text (L266):

“Locust were”

Comments 16: L436 replace triple dot by unique dot

Response 16: We have removed the superfluous punctuation.

Comments 17: L661-L662 please check the correct scientific name of plant specie

Response 17: We have revised the expression.

Revised text (L661-662):

“black locust”

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Comments 1: The sentence "Notably, 37 flavonoids, including flavonols and glycosylated derivatives were consistently upregulated,." ends abruptly with a misplaced comma. It should be revised for better flow.

Response 1: We have removed the extra commas at the end of the sentence to make it more fluent, and add commas after "glycosylated derivatives" to make it more consistent with English expressions.

Revised text (L32-33):

“Notably, 37 flavonoids, including flavonols and glycosylated derivatives, were consistently upregulated.”

Comments 2: You use "moderate drought (40% throughfall reduction)" and "extreme drought (80% throughfall reduction)," but you don’t explicitly state what constitutes "ambient condition." It would be clearer to specify that ambient conditions have 0% throughfall reduction. Also, remove or complete the parenthesis at the end of the sentence.

Response 2: We have revised the expression of control treatment. The inappropriate parentheses have also been removed.

Revised text (L25-26):

“moderate drought (40% throughfall reduction), extreme drought (80% throughfall reduction), and 0% throughfall reduction for control. ”

Comments 3: The phrase "potentially enhancing drought resistance through ROS scavenging and cell membranes stabilization" suggests a hypothesis. Consider clearly stating that further research is needed to confirm this mechanism.

Response 3: Thank you for pointing this out. The increase in flavonoid content may be due to the removal of reactive oxygen species and the stabilization of cell membranes to enhance drought resistance. No verification test was conducted in this experiment, which was deleted because it was deemed inappropriate in the abstract based on the results of previous studies.

Revised text (L33-34):

“These findings suggest that drought induces flavonoid accumulation.”     

Comments 4: "physiological reecological protection strategiessponse" in the keywords appears to be a typographical error. Consider revising it for clarity.

Response 4: Thank you for pointing this out.We have reformatted this part  in the key words to make its meaning more clear.

Revised text (L39):

“Physiological ecology protection strategies”

Comments 5: The phrase "the flavonoid compounds and black locustin coping with drought stress" is unclear. Consider rewording it to "the role of flavonoid compounds in helping Robinia pseudoacacia cope with drought stress."

Response 5: We have modified the original sentence, changing "the response of flavonoids" in the original sentence to "the role of flavonoid compounds in helping", making the expression more accurate and clear, and deleting the redundant "L" in the expression of black locust.

Revised text (L134-136):

“However, the role of flavonoid compounds in helping black locust cope with drought stress has not been reported.”

Comments 6: Some sentences are overly long and could benefit from restructuring to improve readability, such as "The response mechanism of black locust to drought stress has attracted significant attention in regional ecological studies." Please specify the aspects of the response mechanisms that have been studied.

Response 6: We have added specific research aspects to the original sentence to make the expression more specific, and also adjusted the expression of the language to make the expression more fluid.

Revised text (L108-112):

“The degradation of black locust plantations has severely affected the ecosystem stability and ecological functions. In regional ecological studies, specific aspects of black locust's drought stress response mechanisms, such as morphological changes, water-related physiological and biochemical responses, and ecological adaptability, have become research hotspots.”

Comments 7: The introduction could be more logically organized. The discussion on drought stress effects on plants should be introduced before discussing specific impacts on Robinia pseudoacacia.

Response 7: We have adjusted the introduction (L97-112) of robinia acacia to introduce the research progress related to drought stress first, and then related to robinia acacia research to make the paragraphs more coherent.

Comments 8: The transition between sections is sometimes abrupt. For example, the sentence "Water plays a crucial role in the physiological and biochemical processes of plant cells" shifts focus suddenly from afforestation policies to plant physiology. Consider adding a connecting sentence that explains why this transition is relevant.

Response 8: We have revised this part accordingly. In front of the sentence "Water plays a crucial role in the physiological and biochemical processes of plant cells", the important relationship between water and drought is expounded, and a connecting sentence is added to explain and make the paragraphs more coherent. We have also added citation in the revised section.

Revised text (L78-81):

“ Studying the hydrological, physiological, and biochemical responses of mature forests to drought stress can clarify the sensitivity and adaptability of forest systems to drought.Water content is a key factor influencing plant physiology, and the water cycle is a vital component of the biosphere. Water plays a crucial role in the physiological and biochemical processes of plant cells.”

Comments 9: The sentence "Black locust (Robinia pseudoacacia L.), the world's second most extensively planted broadleaved tree species" should include a citation to support this claim.

Response 9: We have added two related citations, i.e., references [23] and [24] in L97-98 in the revised manuscript. .

Comments 10: The claim that "Robinia pseudoacacia accounts for over 90% of the region's total forest cover" should also be supported with a reference.

Response 10: We added four related citations to support this claim (references [25], [26] and [28])

Comments 11: The sentence "Previous studies of Robinia pseudoacacia seedlings have found that..." ends with a full stop after the citation, making it incomplete. This should be revised for clarity and completeness.

Response 11: We have removed the superfluous punctuation marks in the sentences and reformatted the sentences and documents.

Revised text (L114):

“ And previous studies of robinia acacia seedlings have found that under drought stress black locust seedlings can respond rapidly[32–35]. ”

Comments 12: There are several instances where "black locust" is written as "black locustL." or "black locustin." Ensure consistency in formatting.

Response 12: Thank you for pointing this out.We have unified all the expressions of acacia and changed them to “black locust” and “the black locust forest” in the revised manuscript.

Comments 13: There are some formatting errors such as “black locustwas”, please correct them.

Response 13: We have corrected the errors in the format and marked the manuscript in red.

Comments 14: The phrase "ecological functions and the exertion of its ecological functions" is redundant. Consider revising for conciseness.

Response 14: We have rephrased the sentence to make it more concise.

Revised text (L148-151):

“The purpose of this research is to investigate the response of black locust to long-term drought, and to provide scientific basis for screening plant hormones and flavonoids to enhance drought resistance of black locust and to protect regional ecosystem.”

Comments 15: The introduction is overly long and should be more logical and concise.

Response 15: We have reorganized the introduction.

Revised text (L42-151):

Comments 16: Add a supporting reference in section 2.1, while stating average annual temperature and precipitation.

Response 16: We obtained meteorological data based on instruments set up 200 meters away from the experimental sample site, supplementing the meteorological data from July 2022 to August 2023.

Revised text (L164-171):

“Located approximately 200 m away from the study site. The mean precipitation during the observation period (1 July 2022 to 31 August 2023) was 561.51 mm, while the mean surface air temperature recorded was 11.32 °C. Air temperature (Ta) was measured using a temperature sensor (ATMOS-14; METER Group Inc., USA), and precipitation (P) was measured using a tipping bucket rain gauge (6466 m; Davis Instruments, USA)..”

Comments 17: Section 2.2, "Throughfall exclusion were achieved..." Should be "Throughfall exclusion was achieved…", similarly, "Each of the plot covers..." Should be "Each plot covers.

Response 17: We have revised the expression in this section to make the sentence more correct.

Revised text (L179):

“Throughfall exclusion was achieved by adjusting the distance; Each plot covers”

Comments 18: Section 2.2, instead of "please refer to Liu et al. (2024)," consider a more formal phrasing: "Further details on the study site and methodology are available in Liu et al. (2024)."’

Response 18: We have changed the expression to make the language more smooth and grammatically correct..

Revised text (L183-184):

“Further details on the study site and methodology are available in Liu et al. (2024)..”

Comments 19: Section 2.5, The phrase "supernatant was aspirated" is repeated multiple times; rephrase for variation.

Response 19: We have modified the expression "absorb supernatant".

Revised text (L212):

“An appropriate amount of;the clear supernatant was aspirated; the supernatant”

Comments 20: Section “Leaf relative water content” have double full stops in few sentences.

Response 20: We have deleted all parts of the paragraph with double periods, leaving only one period to make the sentence more correct.

Revised text (L212):

“The leaf fresh weight was measured immediately after the sample was collected. ”

Comments 21: Some analytical methods reference studies, but others lack citations. Ensure all methods have proper references.

Response 21: All of our analytical methods are referenced and marked in red.

Comments 22: Proofread for grammar, consistency, and clarity.

Response 22: We have checked and revised the manuscript for grammar, consistency, and clarity.

Comments 23: Ensure uniform terminology and abbreviations (e.g., “HPLC-TC” vs. “HPLCTC”).

Response 23: We have revised this sentence in the manuscript to make it more accurate.

Revised text (L306):

“The high-performance liquid chromatography (HPLC) system combined with a tandem chromatographic separation module was utilized for analysis.”

Comments 24: Provide reference for OriginPro 2022.

Response 24: The reference for Origin 2024 has been provided.

Revised text (L360):

“The graphical representations were created using Origin 2024 (Origin Lab Corporation, Northampton, MA, USA).”

Comments 25: Add a short explanation of heat map analysis and clustering methods in statistical analysis.

Response 25: We added the installation package and parameter Settings for R heat

Revised text (L362-365):

“The heatmap was drawn using the pheatmap package in R language. The parameters were set to retain two decimal places, and row clustering was performed. The horizontal and vertical parameters were set, and then the heatmap was plotted.”

Comments 26: Section 3.1, some sentences are repetitive and can be streamlined for better readability. For example: "No significant differences in the transverse axis length of the guard cells were observed among treatments."  And "Under different treatment conditions, no significant difference was observed in the transverse axis length of guard cells in black locust leaves."

Response 26: We have made this section more concise by removing superfluous sentences.

Revised text (L376-378):

“ which was 49.20% smaller than that in CK. No significant differences in the transverse axis length of the guard cells were observed among treatments. ”

Comments 27: Figures 3, 4, 5 and 8 are hard to read, improve their quality and maximize pixels.

Response 27: We have modified the figures to improve their readability.

Comments 28: Section 3.2.2, The phrase "increase of drought stress" is awkward it should be "as drought stress intensified."

Response 28: We have corrected the misrepresentation.

Revised text (L419):

“as drought stress intensified”

Comments 29: Section 3.3, The sentence "Under ED treatment, the contents of IAA and JA increased by 23.47% and 21.76%, respectively, compared with the control..." is incomplete.

Response 29: We have rephrased this sentence.

Revised text (L448-470):

“The contents of IAA and JA in ED treatment group were increased by 23.47% and 21.76%, respectively. ”

Comments 30: The sentence "The contents of GA-1 showed gradually decrease trend, while GA-3, GA-4, and GA-7 showed increase and decrease trend with the increase of drought level”; Poor phrasing and lacks clarity.

Response 30: We have redescribed this data to make it more complete.

Revised text (L472-474):

“As drought stress intensified,the content of GA-1 showed a gradually decreasing trend, while the contents of GA-3, GA-4 and GA-7 exhibited a fluctuating pattern, increasing first and then decreasing.”

Comments 31: Strengthen scientific interpretation, particularly for oxidative stress, osmolyte accumulation, hormone fluctuations and secondary metabolites.

Response 31: We have revised the Results and Discussion section accordingly to strength the scientific interpretation.

Comments 32:First sentence of section 3.4.1 is overly long and difficult to follow.

Response 32: We have adjusted and streamlined the first sentence to show the specific results of the compounds tested for flavonoids.

Revised text (L491-493):

“Broad-spectrum flavonoid analysis identified 245 distinct compounds classified into 16 subclasses, with specific subclass designations and quantitative distributions detailed. ”

Comments 33:Section 3.4.1, the sentence “Samples within each group were also closely clustered” is Slightly redundant given the previous sentence. Combine for better flow.

Response 33: We have modified the original sentence to make it complete and more concise.

Revised text (L493-495):

“The results of the Principal Component Analysis (PCA) indicated that the Quality Control (QC) samples were highly clustered, suggesting good sample repeatability and high detection quality. There were obvious separations among the various experimental groups.”

Comments 34:Do not need to mention “show this” in captions of figure 5 & 6.

Response 34: We have removed the term “show this” from Figures 5 and 6

Comments 35:Section 3.4.2, The manuscript states "Based on the results of OPLS-DA, with VIP>1 and p<0.05 as the criteria, differential metabolites (DEMs) were screened for comparison groups,” While VIP > 1 is a common criterion for selecting important metabolites, it is important to clarify how p-values were adjusted for multiple comparisons. Briefly mention whether multiple testing corrections (False Discovery Rate (FDR) or Bonferroni applied) were performed to avoid false positives.

Response 35: We consulted the data report of the company's testing, and obtained the number of OPLS-DA replacement tests: 200, which was corrected to ensure the accuracy of the data.

Revised text (L517-519):

“The number of replacement tests in OPLS-DA analysis was 200, and P value<0.05 of T-test was corrected.”

Comments 36:Table 2-5 should be written as S1-S5 within text to avoid any confusion.

Response 36: The table number has been revised.

Revised text (L102):

“TableS1-S4”

Comments 37: The discussion lacks a clear flow between sections. Some sub-sections are well-structured, but transitions between physiological responses, osmotic adjustment, and metabolomics findings need better coherence.

Response 37: We have revised the Discussion section to avoid any misunderstanding.

Revised text (L566-738):

Comments 38:The section headings (e.g., “3.2 Effects of Drought Stress on Leaf Physiology of Robinia pseudoacacia”) should be correctly numbered to maintain consistency with the rest of the manuscript.

Response 38: We have revised section heading.

Revised text (L411):

“3.2. Effects of Drought Stress on Leaf Physiology of Black Locust ”

Comments 39:The discussion mentions previous studies but does not cite specific references (e.g., "Previous study on the same study site found that under drought stress, black locust exhibited reduced transpiration rate and gas exchange rate..."). Provide citations.

Response 39: We have added relevant references [29], [61] to the statements in the revised manuscript.

Comments 40: Section 4.2, the RWC trends should be compared quantitatively.  Instead of "exhibited a progressive decline," specify the percentage decrease observed under MD and ED conditions.

Response 40: Experimental data have been added to the statement to make it more specific.

Revised text (L612):

“The studies revealed that The RWC of MD and ED was 10.39% and 14.69% lower than that of CK (68.61%). ”

Comments 41:The phrase, "Metabolomics technology enables us to obtain qualitative and quantitative information of numerous metabolites simultaneously," is ambiguous. Instead, briefly describe the analytical approach (e.g., LC-MS/MS).

Response 41: We have revised the expression of this sentence to make it more complete and accurate

Revised text (L731-732):

“LC-MS/MS metabolomics technology enables us to obtain qualitative and quantitative information about a variety of metabolites.”

Comments 42:Some results, such as ABA trends and MDA accumulation, deviate from expected physiological responses. Provide alternative explanations or cite supporting literature.

Response 42: We have added relevant references [39], [84] [90] and [91] to the statements in the revised manuscript.

Comments 43:The phrase "indicating the species may approach its drought-resistance threshold" needs more specificity. What physiological indicators (e.g., MDA accumulation, hormonal imbalances) suggest this threshold?

Response 43: Thank you for pointing this out. We agree with this comment.The values of MDA, proline, osmoregulatory substances and hormones can be explained.

Comments 44:Since flavonoid metabolomics was a major component of the study, briefly mention how specific flavonoids contributed to drought resistance (e.g., ROS scavenging, osmotic balance).

Response 44: Thank you for pointing this out. We agree with this comment.The mechanism of flavonoid metabolome related to drought resistance is described in this paper, which makes it more clear and specific.

Revised text (L125-137):

“Flavonoids directly neutralize reactive oxygen species (ROS) through their phenolic hydroxyl groups and maintaining cell membrane integrity through lipid peroxidation inhibitiongHowever, the role of flavonoid compounds in helping black locust cope with drought stress has not been reported.”

Comments 45:Some references do not adhere to the journal's format, with missing volume and page numbers. Please ensure these details are correctly included.

Response 45: We have checked and corrected the format of the references in the revised manuscript.

Comments 46:In supplementary material there is no need to write “this is the table”, just write table 1, 2 etc.,

Response 46: We have corrected the language used in the supplementary materials.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Comments 1: Line 73. Please provide supporting references to this proposal.

Response 1: TWe have added the relevant literature to support this statement (i.e., reference [30], [31])

Comments 2: Line 107-109. Put the sentence in order and place the references correctly.

Response 2: We have revised this part accordingly.

Revised text (L91-93):

“These molecular responses involve complex metabolic networks that produce intermediates and end-products functioning as regulatory and signaling molecules in the plant's adaptive mechanisms.”

Comments 3: Line 167. “refer to Liu et al., 2024” - indicate the reference number in the list of references.

Comments 4: Line 170-171. "intact sunny leaves were collected from three trees of similar age and growth status" - please specify the age of the trees from which the plant material was collected.

Response 4: We have pointed out in the original article that the age of the black locust trees in the sample is 20 years.

“The throughfall exclusion experiment was conducted in a 20-year-old black locust plantation.”

Comments 5: Line 208. "according to the following formulas" - I did not find the necessary formulas after following the link. Please insert the formulas into the article showing how the calculations were made.

Response 5: We have supplemented the calculation formula in L226-230.

Comments 6: Line 270. List all eight hormones measured.

Response 6: We supplemented all eight of the hormones tested in the experiment.Revised text (L294-297):

“The concentrations of eight hormones, including gibberellic acid (GA1, GA3, GA4, GA7), jasmonic acid (JA), salicylic acid (SA), indoleacetic acid (IAA), and abscisic acid (ABA), ”

Comments 7: Figure 2. A and B are not mentioned in the text of the article.

Response 7:  Figure 2A and 2B were referred in L391.

Revised text (L391):

“Under normal moisture conditions, the epidermal hairs on the lower epidermis of the leaves were arranged relatively sparsely, but their quantity was higher than that on the upper epidermis (Figure. 2A, 2B 2C and 2D).”

Comments 8: Line 436 and 627 are extra dots.

Response 8: We have removed the superfluous punctuation.

Comments 9: In the text of the article, you need to describe the gibberellins whose levels you measured. Explain what GA-1, GA-3, GA-4, GA-7 are.

Response 9:  We further explained the role of GA1, GA3, GA4 and GA7 in plant growth in the discussion section 4.3..

Revised text (L684-690):

“As of 2022, 136 gibberellins (GAs) have been identified, with GA1, GA3, GA4, and GA7 being the free-active forms. Current research indicates that only these four bioactive GAs possess biological activity, while most other GA species do not. GA1 and GA3 primarily regulate plant growth and development, and upon drought stress, they can scavenge excess ROS to mitigate the stress impact on plant growth. GA4 promotes fruit maturation and leaf senescence, while GA7 mainly enhances plant abiotic stress tolerance.”

Comments 10: Figures 5 and 6. The caption under the figure needs to be corrected. Remove the word "shows".

Response 10: The figure caption has been revised.

Comments 11: Line 475. Why did you indicate “can be further analyzed” and not perform the analysis yourself?

Response 11: The OPLS-DA and PCA analyses demonstrated significant discrimination among the three groups, making it feasible to conduct further analysis on the up - or down - regulation of flavonoid content in drought - stress conditions. This sentence has been modified to avoid misunderstanding.

Revised text (L506):

“The difference of flavonoids was further analyzed”

Comments 12: Line 545. "Previous study on the same study site" - provide a supporting link.

Response 12: We have reformulated this sentence and added related research literature based on your and other reviewers [30 62].

Comments 13: Line 561. Numbering is mixed up. Correct 3.2 to 4.2

Response 13: We have changed the heading numbers to make sure they are in the correct order.

Comments 14: Line 568. "This finding aligns with those of other relevant investigations" - with the results of what relevant investigations? Provide references

Response 14: We have added relevant references([23] [33]) studying on black locust seedlings, which found a decreased RWC with the strengthening of drought stress, similar to the results found in this study.

Comments 15: Line 570. "Previous studies…". What studies? Provide references.

Response 15:  We annotated the relevant literature [94].

Comments 16: Line 583. "which is consistent with the results of previous studies" - With which studies? Provide references.

Response 16: We later note the references, which are partly based on the results of Dauri Hu Zhiko's research [64,65].

Comments 17: Line 604-605: Provide a supporting reference in this sentence.

Response 17: We inserted the relevant literature in this sentence [68,70].

Comments 18: Line 650-653. "This trend aligns with previous findings" - whose previous findings? Reference needed.

Response 18: We have added relevant literature annotations to this paragraph[79].

Comments 19: Line 661. “The results of previous studies” - provide a reference to this work.

Response 19: We have added relevant literature annotations to this paragraph[72].

Comments 20: Line 666-668. Link 74. I could not find an English version of this article. Judging by the abstract and title I read, it is about the content of endogenous ABA and there is no mention of flavonoids. Please double check.

Response 20: We checked that there were some problems with the placement of the literature, and we changed the correct flavonoid literature and made adjustments to this literature[94].

Comments 20: Overall, there are many comments, but none of them are major. I would also like to note that the supplementary materials look out of place in your article. Please move them to the text of your article. Supplement the methods in the article and move all the tables to the article, inserting them as they are mentioned in the text.

Response 21: In this study, the physiological and biochemical responses of black locust to drought stress were discussed by exploring the physiological and biochemical level of leaf microstructure. Our conclusion is to summarize the results of the study.We have adjusted the table to the body.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Comments 1: The title of the manuscript is pertinent; however, it emphasizes flavonoids, but the study also investigates other compounds, such as osmolytes and hormones. If the goal is to cover the entire metabolism of the plant under stress, it could include "biochemical responses" instead of being specific.

Response 1: This study focuses on both physiological and flavonoids metabolic responses of black locust leaves to drought stress. Osmoregulatory substances and hormones are attributed to plant physiology. The current title represents our dual focus on physiological adaptation and flavonoid metabolic regulation.

Comments 2: Excessively long and repetitive opening sentence, see: “Drought severely affects the stability and ecological functions of artificial Robinia pseudoacacia forests on the Loess Plateau, yet there is limited research on the physiological and biochemical responses of mature black locust to drought stress”.

_ The sentence contains redundant ideas. The impact of drought on ecological stability already implies the need to study its physiological and biochemical responses.

_ "Physiological and biochemical responses" are mentioned, but later on the abstract focuses only on "physiological and metabolic responses", causing inconsistency.

Response 2: We have simplified the beginning of the abstract and unified the expression, changing it to "physiological and metabolic responses. "

Revised text (L20-23):

“Drought threatens the stability of artificial Robinia pseudoacacia forests on the Loess Plateau, yet there is limited research on the physiological and metabolic responses of mature black locust to drought stress. ”

Comments 3: b. Description of the experiment is difficult to follow, see: “This study investigated the leaf microstructure, relative water content (RWC), osmotic adjustment substances, hormone levels, and flavonoid metabolites in black locust under different levels of drought stress using a throughfall exclusion experiment (i.e., moderate drought (40% throughfall reduction), extreme drought (80% throughfall reduction), and ambient condition with no reduction).”

_ "Different levels of drought stress" is vague and could be omitted, since the drought levels are already specified.

_ "Throughfall exclusion experiment" is a technical term that could be clearer.

_ The sentence is long and difficult to process.

Response 3: We have simplified the long sentences in the experimental design part, and revised the contents to be more clear.

Revised text (L22-23):

“This study employed a throughfall exclusion system (i.e., moderate drought (40% throughfall reduction), extreme drought (80% throughfall reduction), and 0% throughfall reduction for control.) to analyze leaf microstructure, relative water content (RWC), osmotic adjustment substances, hormone levels, and flavonoid metabolites in black locust under controlled drought stress.”

Comments 4: c. Transition problem in the results, see: “Results showed that as drought stress intensified, stomatal aperture and density decreased progressively, whereas trichome density and length significantly increased.”

_ "Decreased progressively" is unnecessary, since "decreased" already indicates a continuous change.

_ "Whereas" suggests a contrast, but both phenomena are part of the plant's adaptation to drought.

Response 4: We further refined and modified the expressions to make the sentences more concise and correct.

Revised text (L27-29):

“The results demonstrated that as drought stress intensified, stomatal aperture and density decreased, while trichome density and length exhibited significant increases.”

Comments 5: d. For scientific accuracy and agreement, see: RWC, photosynthetic pigments, and ABA content declined significantly, whereas MDA, proline, IAA, and osmotic adjustment substances initially increased with drought stress but decreased under extreme drought.

_ "Osmotic adjustment substances" is vague; the reader does not know which specific substances were analyzed.

_ "Initially increased with drought stress but decreased under extreme drought" could be clearer and more concise.

Response 5: We have revised the expression to make it more clear and correct.

Revised text (L29-30):

“MDA, proline, IAA, and osmotic adjustment substances (soluble protein, reducing sugar, and total sugar) first increased and then decreased as drought stress intensified.”

Comments 6: e. Punctuation and structure problem in metabolomics, see: “Metabolomic analysis identified 245 flavonoid compounds, with 91 differentially expressed metabolites under drought treatments. Notably, 37 flavonoids, including flavonols and glycosylated derivatives were consistently upregulated,.”

_ "Metabolomic analysis identified 245 flavonoid compounds", the sentence could be more objective.

_ "Flavonols and glycosylated derivatives" needs a comma before "were".

_ The period is incorrect.

Response 6: We have modified the sentences to make them more concise and ensure the unity of tenses.

Revised text (L31-34):

“A total of 245 flavonoid compounds were identified through metabolomic analysis, among which 91 exhibited differential expression under drought treatments. ”

Comments 7: f. Scientific accuracy problem, see: “These findings suggest that drought induces flavonoid accumulation, potentially enhancing drought resistance through ROS scavenging and cell membranes stabilization.”

_ "Drought induces flavonoid accumulation" is too straightforward for an observational study. It could be reworded to reflect the correlational nature.

_ "Cell membranes stabilization" → the correct form would be "cell membrane stabilization" (singular).

Response 7: This paragraph has been condensed in accordance with your and other reviewers' comments, making the sentences more accurate and the words more correctly spelled.

Revised text (L33-34):

“These findings suggest that drought stress can lead to the accumulation of flavonoids.”

Comments 8: g. Overgeneralization in the conclusion, see: “This study elucidates the physiological and metabolic responses of mature black locust to drought stress, providing theoretical support for vegetation restoration and ecological management strategies in arid regions.”

_ "Providing theoretical support" is vague. Does the study offer practical implications?

_ "Vegetation restoration and ecological management" are broad terms.

Response 8: The purpose of the study has been modified to make the expression more clear and correct.

Revised text (L34-37):

“This study explores the physiological and metabolic responses of mature black locust trees to drought stress, offering insights for selecting drought-resistant species in vegetation restoration and informing ecological management practices in arid regions.”

Comments 9: a. The introduction contextualizes the problem well, but does not present a clear hypothesis or central scientific question. E.g., “Understanding the physiological and ecological responses of mature black locust forests to drought stress is of great significance for forest management, ecological restoration, and predicting the response of forest ecosystems to climate change.” This sentence explains the importance of the study, but does not clearly define what is being tested or what specific gap will be filled. Please explicitly add a hypothesis or central question in the introduction.

Response 9: We elaborated on the purpose of the study to make the sentence more clear.

Revised text (L124-127):

“Understanding the responses of mature Robinia pseudoacacia (black locust) forests to drought stress in terms of leaf microstructure, physiology, and flavonoid compounds is critical for forest management, ecological restoration, and predicting forest ecosystem responses to climate change.”

Comments 10: b. The introduction mentions flavonoids, but does not clearly explain their role in the response to drought stress.

Response 10: The reasons for the response of flavonoids to drought stress were described to make the description more complete and accurate.

Revised text (L134-135):

“Flavonoids directly neutralize reactive oxygen species (ROS) through their phenolic hydroxyl groups and maintaining cell membrane integrity through lipid peroxidation inhibition.”

Comments 11: c. Rephrase the objective to make it more specific and aligned with the expected results. See this example: “This study investigates how drought stress influences leaf morphology, water balance, hormone regulation, and flavonoid metabolism in mature black locust (Robinia pseudoacacia) plantations. By integrating physiological, biochemical, and metabolomic analyses, we aim to identify key drought adaptation mechanisms and their implications for forest management in arid regions.”

Response 11: We have revised the research purpose in the introduction to make the expression more clear and accurate.

Revised text (L149-152):

“ The purpose of this research is to investigate the response of black locust to long-term drought, and to provide scientific basis for screening plant hormones and flavonoids to enhance drought resistance of black locust and to protect regional ecosystem.”

Comments 12: a. There is no clear description of the environmental conditions of the experiment other than the drought treatment.

Response 12: In the experimental method, we introduce the geomorphology and annual precipitation of Wuqi, Shaanxi Province, as well as the specific layout and detection method of the experimental plot. It contains all aspects related to the experiment.

Revised text (L159-185)

Comments 13: b. The study does not mention variations in temperature, soil moisture, or other environmental factors that could influence the results.

Response 13: We added the relevant values of soil water content and annual mean temperature to make the expression more accurate and clear.

Revised text (L165-172):

“Located approximately 200 m away from the study site. The mean precipitation during the observation period (1 July 2022 to 31 August 2023) was 1.50 mm, while the mean surface air temperature recorded was 11.32 °C. Air temperature (Ta) was measured using a temperature sensor (ATMOS-14; METER Group Inc., USA), and precipitation (P) was measured using a tipping bucket rain gauge (6466 m; Davis Instruments, USA).The soil water content of the sample plots was measured by laying pipes directly from 20cm to 500cm, and the average soil water content was 6.36Avg from January 2023 to September 2023.

Comments 14: c. How was it ensured that the exclusion of rainfall did not impact other environmental factors, such as leaf temperature and transpiration? Was the excluded water diverted away from the roots or infiltrated into the nearby soil?

_ The authors should: Indicate whether additional environmental conditions were measured (e.g., soil temperature, relative humidity, evapotranspiration). AND Explain whether the plots were randomized to avoid environmental bias.

Response 14: The comments are not directly replied.(L183-186)

1. Admit that the exclusion of rainfall would have affect other environmental factors such as soil temperature, leaf temperature, but we did not measure these factors.

2. excluded water were discharged away from the plot.

3. plots were randomly placed in the study site.

4. for more detail, refer to Liu et al. (2024).

Comments 15: d. The study mentions that three plots were used per treatment, with approximately 60 trees per plot. However, it is not clear how many leaves or trees were actually used for each measurement.

_ State how many trees were actually sampled in each plot.

_ Explain how many leaves were collected per tree for each type of analysis.

_ If samples were pooled, indicate this clearly.

Response 15: We added the specific information of sample collection, and selected three trees in each sample plot to collect a total of 80 leaves for follow-up experiments.

Revised text (L185-188):

“In each plot, 3 trees were selected, with 20 - 30 leaves collected from each tree, resulting in a total of 80 leaves. The leaves were then mixed together to facilitate subsequent experiments.”

Comments 16: e. The study mentions that ANOVA was used to compare groups, but does not specify:

_ Whether the data were tested for normality and homogeneity of variance before ANOVA.

_ Whether any multiple comparison method was applied (Tukey, Bonferroni, Duncan, etc.).

_ Whether the analysis was done by tree individually or with averages per plot.

Response 16: The data were tested for normality and homogeneity of variance.

Multivariate comparison was not used, single factor analysis of variance was performed, and the analysis was conducted by block average.(L369-372)

Comments 17: a. "No significant differences in the transverse axis length of the guard cells were observed among treatments. Under different treatment conditions, no significant difference was observed in the transverse axis length of guard cells in black locust leaves.". This sentence repeats the same information twice. It could be rewritten more concisely.

Response 17: We have made this section more concise by removing superfluous sentences

Revised text (L378-380):

“ No significant differences in the transverse axis length of the guard cells were observed among treatments. ”

Comments 18: b. "Under normal moisture conditions, the epidermal hairs on the lower epidermis of the leaves were arranged relatively sparsely, but their quantity was higher than that on the upper epidermis (Figure. 2C and 2D).". The use of "relatively sparsely" is unnatural. "Sparsely arranged" already conveys the idea of sparse distribution.

Response 18: We have revised the expression to make it more precise and clear.

Revised text (L390-392):

“Under normal moisture conditions, the epidermal hairs on the lower epidermis of the leaves were arranged sparsely arranged, but their quantity was higher than that on the upper epidermis (Figure. 2A, 2B 2C and 2D).”

Comments 19: c. "Besides, in ED treatment, the length of the stomata was significantly reduced, with an average of 3.05 µm, which was 49.20% smaller than that in CK.". Instead of "The length of the stomata was significantly reduced" change it to "stomatal length significantly decreased" for greater objectivity.

Response 19: We have modified the wording to make the expression more perfect and accurate.

Revised text (L380):

“stomatal length significantly decreased”

Comments 20: d. "Relative water content (RWC) of leaves serves as a pivotal metric for assessing the water status and drought-tolerance capabilities of plants.". "Capabilities of plants" is not necessary; "drought tolerance" already conveys that idea.

Response 20: According to your suggestions, we have removed the inappropriate words to make the sentences more concise and accurate.

Revised text (L602-604):

“Relative water content (RWC) of leaves serves as a pivotal metric for assessing the water status and drought - tolerance ”

Comments 21: e. "The MDA contents under MD and ED were 28.17% and 9.04% higher than that under CK, respectively.". "Contents" is not the best term to refer to the level of a compound. "Levels" is more accurate.

Response 21:  According to your suggestion, we have changed the inappropriate words to make the sentence more concise and accurate.

Revised text (L434-435):

“The MDA levels under MD and ED were 28.17% and 9.04% higher than that under CK, respectively. ”

Comments 22: f. "As drought stress intensified, the stomata on the lower epidermis gradually closed, transitioning from an open to a closed state, indicated by reduced stomatal widths in both MD and ED treatments compared to that in CK.". Stomatal closure can be caused by multiple factors, including loss of guard cell turgor and hormonal regulation. The text assumes that stomatal closure is a direct reflection of stress intensity, but does not mention whether this response was confirmed by physiological measurements such as osmotic pressure or relative water content.

Response 22: In this study, we tested the support of RWC for the relative water content of leaves. Stomatal closure in this study is a direct reflection of stress intensity.

Comments 23: g. "The MDA contents under drought treatments was significantly higher than that under CK (p<0.05), with the highest MDA content found under MD. [...] Notably, MDA contents were higher under MD than ED treatment, suggesting that the physiological response to drought may have approached its threshold.". Here there is a possible contradiction. If MDA (an indicator of oxidative stress) was higher under MD than under ED, this may indicate that under extreme stress (ED), the plant may have reduced its metabolic functions to the point of minimizing MDA generation, rather than suggesting that the stress response has reached a "threshold". In many cases, a lower level of MDA under extreme drought may indicate cellular collapse or loss of stress response capacity.

Response 23: Our presentation of the MDA-related results in the discussion section below has been revised in response to your comments to make it more accurate.

Revised text (L643-645):

“This may indicate that under ED treatment, Robinia pseudoacacia could have reduced its metabolic functions to minimize MDA production, suggesting that its cells might have collapsed or lost their stress - response capacity. ”

Comments 24: h. "In response to drought, the abscisic acid (ABA) content in black locust leaves showed a gradually decreasing trend.". The literature generally indicates that ABA increases in response to drought stress, as it is one of the main hormones responsible for stomatal closure and adaptation to water deficit. The decrease in ABA with increasing drought may indicate distinct mechanisms, such as hormonal degradation or a species-specific response. The text does not discuss alternatives for this unexpected response.

Response 24: In the following discussion, we discussed the reasons for the decrease of ABA content in recent years.

Revised text (L704-709):

“In the present study, the content of abscisic acid (ABA) gradually decreased with the increasing degree of drought stress. Accumulating evidence indicates that as drought stress persists, genes responsible for ABA biosynthesis are down - regulated, while those involved in its catabolism are up - regulated. This over - activation disrupts the normal regulation of ABA metabolism, ultimately culminating in a reduction of ABA content. ”

Comments 25: i. "Under drought stress, 37 flavonoid-related substances in black locust were continuously upregulated to enhance its drought resistance.". The sentence implies that the upregulation of flavonoids causes greater resistance to drought, when in fact it is a correlation. Although there is evidence that flavonoids act as antioxidants and may be involved in the response to drought stress, the study does not demonstrate causality.

Response 25: According to your suggestion, we have modified this statement to make it more correct and complete.

Revised text (L721-723):

“When under drought stress, 37 flavonoid - related substances in black locust were continuously upregulated , and 14 of them showed significant differences in three comparison groups”

Comments 26: j. I was unable to analyze Figure 8 properly. The board and captions were too small.

Response 26: We have changed the picture to improve the readability.

“Figure 8”

Comments 27: a. "These responses help black locust reduce water loss, thereby strengthening its ability to withstand drought.". The statement assumes that increased trichome density causes increased drought resistance, but there is no direct test of this relationship in the study. Although this is a valid hypothesis, it should be presented with more caution.

Response 27: We refined the discussion to make it more accurate.

Revised text (L599-602):

“These reactions are considered to help black locust reduce water loss, thereby enhancing its drought resistance. However, further research is needed to confirm the correlation between trichomes and drought resistance. ”

Comments 28: b. "When plants are subjected to drought stress, the closure of stomata leads to the restriction of carbon dioxide supply, which directly inhibits photosynthesis or carbon metabolism, resulting in the decrease of photosynthetic pigment content.". This statement is valid, but it is an overgeneralization. Not all plants respond in the same way, and the reduction in photosynthetic rate may depend on the severity of the drought, the species, and additional physiological factors.

Response 28: We have revised this paragraph according to your suggestion to make the sentence more accurate.

Revised text (L625-629):

“When plants are subjected to drought stress, the closure of stomata restricts carbon dioxide supply, directly inhibiting photosynthesis or carbon metabolism and causing a decrease in photosynthetic pigment content. However, different plants respond differently to drought stress due to its severity and other physiological factors. ”

Comments 29: c. "In the present study, the content of abscisic acid (ABA) gradually decreased with the increasing degree of drought stress.". Typically, ABA increases under drought stress to induce stomatal closure and other physiological responses. If ABA levels were reduced under extreme drought, it may be due to a breakdown of the synthesis pathway, accelerated degradation, or hormonal redistribution.

Response 29: We have made changes based on your comments and those of other reviewers to make the sentences more accurate.

Revised text (L704-710):

“In the present study, the content of abscisic acid (ABA) gradually decreased with the increasing degree of drought stress. Accumulating evidence indicates that as drought stress persists, genes responsible for ABA biosynthesis are down - regulated, while those involved in its catabolism are up - regulated. This over - activation disrupts the normal regulation of ABA metabolism, ultimately culminating in a reduction of ABA content[90].”

Comments 30: d. "The MDA levels in drought-stressed black locust leaves were significantly increased compared to that in CK, indicating that the degree of lipid peroxidation of cell membranes was aggravated. Notably, MDA contents were higher under MD than ED treatment, suggesting that the physiological response to drought may have approached its threshold.". As mentioned earlier in the analysis of the results, if MDA was higher under moderate drought (MD) than under extreme drought (ED), this may indicate that the plant metabolism collapsed under ED, resulting in lower MDA production.

Response 30:

Revised text (L642-645):

“This may indicate that under ED treatment, Robinia pseudoacacia could have reduced its metabolic functions to minimize MDA production, suggesting that its cells might have collapsed or lost their stress - response capacity..”

Comments 31: e. "These results indicate that black locust has unique metabolic characteristics under drought stress.  The identified flavonoids may help scavenge ROS (reactive oxygen species) effectively, thus enhancing its drought resistance.".  Although flavonoids are known for their antioxidant properties, the claim that they enhance drought resistance lacks direct experimental evidence.  The study identified differentially expressed flavonoids but did not directly test their function.

Response 31:  According to your comments, we have revised this sentence to make it more concise and accurate.

Revised text (L602-604):

“These results indicate that black locust has unique metabolic characteristics under drought stress. thus enhancing its drought resistance.

Comments 32: a. The conclusion extrapolates the results to ecological management without providing a clear link to the study findings. How do the study data specifically impact ecological restoration? Are there any practical recommendations?

Response 32: We have further modified and improved the conclusion to make it more accurate and easier for readers to understand.

Revised text (L749-757):

“By studying black locust's leaf microstructure, physiology, and flavonoid metabolomics responses to drought stress, this paper clarifies the specific physiological and biochemical responses of black locust under drought stress. Future research should integrate multi - omics data to explain black locust's drought - resistance mechanisms at the molecular level. Also, long - term field observations combined with climate models are needed. This provides dynamic threshold warnings for sustainable management of mature black locust plantations, supporting the building of ecological barriers in arid areas. ”

4. Response to Comments on the Quality of English Language

Point 1: Example 1: (Abstract, line 13)

"whereas trichome density and length significantly increased.". "Significantly increased" is less natural than "increased significantly" in academic English, since adverbs usually appear after the verb.

Response 1: We have modified it according to your suggestion and marked it in red.

Point 2: Example 2: (Abstract, line 16)

"Notably, 37 flavonoids, including flavonols and glycosylated derivatives were consistently upregulated,.". A comma should be added to separate the subject from the predicate and the extra period at the end of the sentence should be removed.

Response 2: We have modified it according to your suggestion and marked it in red.

“Notably, 37 flavonoids, including flavonols and glycosylated derivatives, were consistently upregulated.”

Point 3: Example 3: (Introduction, line 45)

"The condensed planning density and high water-consuming species have led to the overconsumption of limited soil water resources, resulting in the exceeding of the water carrying capacity of the plantation ecosystem in the region." Change "Condensed planning density" to "high planting density" (more natural); change "High water-consuming species" to "water-demanding species" (more common expression); change "Overconsumption of limited soil water resources" to "excessive soil water consumption" (more direct); and change "Resulting in the exceeding of" to "surpassing" (more concise form).

Response 3: We have modified it according to your suggestion and marked it in red.

“ The high planting density and water-demanding species have led to the excessive soil water consumption water resources, surpassing of the water carrying capacity of the plantation ecosystem in the region”

Point 4: Example 4: (Introduction, line 57)

"The mean annual precipitation of 424mm, mainly occurring from June to September.". The article "the" before "mean annual precipitation" is correct, but the sentence lacked a main verb ("is").

Response 4: We have modified it according to your suggestion and marked it in red.

“The mean annual precipitation is 424mm, mainly occurring from June to September.”

Point 5: Example 5: (Methods, line 110)

"For detailed information of the study site and the throughfall exclusion experiment, please refer to Liu et al (2024).". Change "Information of" to "Information on" (correct use of the preposition).

Response 5: In accordance with your comments and those of other editors, this sentence has now been removed from the article and has been written elsewhere to further elaborate.

Point 6: Example 6: (Results, line 150)

"Significant decline in the relative water content (RWC) of black locust leaves were observed as drought stress intensifies (p<0.05). Change "Significant decline" to "A significant decline" (the noun "decline" needs an article); change "Were observed" to "Was observed" (verb agreement, since "decline" is singular); and change "Intensifies" to "Intensified" (correct verb tense to maintain consistency with "was observed").

Response 6: We have modified it according to your suggestion and marked it in red.

“A significant decline in the relative water content (RWC) of black locust leaves was observed as drought stress intensified”

Point 7: Example 7: (Discussion, line 230)

"Plants have evolved a multi-level defense system to combat drought stress, operating through morphological, physiological, and molecular adaptations.". "Multi-layered defense system" (more common in the scientific literature) and "Incorporating" (better integration with the rest of the sentence).

Response 7: We have modified it according to your suggestion and marked it in red.

“Plants have evolved a multi-level defense system to incorporating drought stress, operating through morphological, physiological, and molecular adaptations.”

Comments 1:In abstract, line 23, remove parenthesis.

Response 1: We made the sentence more complete by removing the superfluous symbols.

Revised text (L23-27):

“This study employed a throughfall exclusion system i.e., moderate drought (40% throughfall reduction), extreme drought (80% throughfall reduction), and 0% throughfall reduction for control to analyze leaf microstructure, relative water content (RWC), osmotic adjustment substances, hormone levels, and flavonoid metabolites in black locust under controlled drought stress.”

Comments 2: In introduction, line 135, there is no need to refer to methodology here.

Response 2: We have deleted the inappropriate part to make the sentence more complete and accurate.

Revised text (L135)

Comments 3: Section 2.1 and 2.2 have formatting errors, please correct them.

Response 3: We adjusted the format to make the presentation more uniform and accurate.

Comments 4: Section 2.3, use capital letters in the start of a sentence. 

Response 4: We adjusted the uncapitalized letters to make the sentences more accurate.

Revised text (L187-188)

“Leaves collected from each tree, resulting in a total of 80 leaves. ”

Comments 5: Figure 4 quality is still low and all the components are not in the same format.

Response 5: We split the image into Figure 4 and Figure 5, and adjusted the following images in turn.

Figure 4 and Figure 5

Comments 6: The Majority of the references do not adhere to journal guidelines.

Response 6: We have adjusted the format of the references.

4. Response to Comments on the Quality of English Language

Point 1:The English is fine and does not require any improvement.

5. Additional clarifications

Please add Prof. Chao Yue's contribution to this manuscript in the Author

Contributions.

Response :We have added teacher Yue Chao's contribution at the end of the article.

“Chao Yue: provide research ideas, situation analysis, data interpretation and scientific research support. ”