Comparative Morpho-Physiological and Biochemical Responses of Waterleaf (Talinum fruticosum) to Drought and Waterlogging Stresses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study evaluates the morphological, physiological, and biochemical responses of waterleaf under two water stress conditions: drought and waterlogging. The authors have clearly justified the need for the study and have presented their results with appropriate statistical analyses. However, the manuscript has several major flaws that need to be addressed before it can be considered for publication. I have listed my comments below for the authors' consideration.

Lines 52–53: I suggest revising the text to “…underutilized, neglected, or orphan leafy vegetables…”.

Lines 82–83: I suggest deleting these lines, as the same information is repeated in Lines 109–111.

Materials and Methods (Lines 164–165): It is not clear whether irrigation was completely withheld for only one week, or whether, after one week, plants were irrigated only to maintain <10% field capacity (FC). Please clarify the experimental procedure.

Lines 170–171 and 184: It is also unclear whether the leaf samples and other observations were collected seven additional days after the initial one-week water withholding period. Please clarify the sampling timeline.

Lines 250–251: The authors state that the figures show standard error (SE), whereas all figure captions indicate that the error bars represent standard deviation (SD). Please resolve this inconsistency.

Results: The scales of a figures do not appear to match the data described in the text. This issue is observed in all panels of Figure 1, all panels of Figure 2 except the Control in panel D, and all panels of Figure 3 except the Control bar in panel A. All figures should be carefully checked and revised so that the graphical scales accurately reflect the values reported in the text.

Line 311: Please correct “chlorophyll a” to “chlorophyll-a.”

Discussion (Lines 477–479): The authors state that the chlorophyll a/b ratio remained stable across treatments. However, no data or figure is provided to support this statement. Please provide supporting results or revise the discussion accordingly.

 

Author Response

Reviewer 1

We would like to express our sincere gratitude for the time and effort you have dedicated to reviewing our manuscript. Your critical and insightful comments have significantly enhanced the quality and scientific rigor of our work. We greatly appreciate your thoughtful suggestions and constructive feedback, which have helped us to substantially improve the manuscript.

We have carefully addressed all of your comments and provided detailed responses below. All revisions have been highlighted in the revised manuscript for easy identification.

1: Lines 52–53: I suggest revising the text to “…underutilized, neglected, or orphan leafy vegetables…”.

Authors’ Response: We thank the reviewer for this constructive suggestion. We have revised the text to read 'underutilized, neglected, or orphan plant species' to better reflect the current terminology in the literature. This change has been made at Lines 52–53 of the revised manuscript.

2: Lines 82–83: I suggest deleting these lines, as the same information is repeated in Lines 109–111.

Authors’ Response: We thank the reviewer for identifying this redundancy. We have deleted Lines 82–83 as suggested, as the identical sentence appears later in the Introduction (Lines 109–111). The revised manuscript now avoids unnecessary repetition."

3: Materials and Methods (Lines 164–165): It is not clear whether irrigation was completely withheld for only one week, or whether, after one week, plants were irrigated only to maintain <10% field capacity (FC). Please clarify the experimental procedure.

Authors’ Response: We have revised the text in Section 2.2 (Lines 164–165) to clarify that drought stress was induced by completely withholding water for seven days, with measurements taken on day 7 when wilting and ≤10% FC were observed. No further irrigation was applied during the stress period. This has been corrected in the revised manuscript.

4: Lines 170–171 and 184: It is also unclear whether the leaf samples and other observations were collected seven additional days after the initial one-week water withholding period. Please clarify the sampling timeline.

Authors’ Response: We thank the reviewer for this important clarification. We have revised Sections 2.3 and 2.4.1 to explicitly state that all measurements and sampling were performed on the same seventh day of the stress treatment—i.e., the day when drought-stressed plants reached ≤10% FC and exhibited visible wilting. No additional waiting period occurred after the initial 7-day water withholding. The revised text now clearly aligns the sampling timeline with the stress induction protocol.

5: Lines 250–251: The authors state that the figures show standard error (SE), whereas all figure captions indicate that the error bars represent standard deviation (SD). Please resolve this inconsistency.

Authors’ Response: We thank the reviewer for identifying this inconsistency. We have corrected Section 2.5 (Lines 250–251) to state that results are presented as means accompanied by standard deviations (SD), which now aligns with all figure captions. The error bars in all figures indeed represent ± SD (n = 5), and this has been consistently reflected throughout the revised manuscript.

6: Results: The scales of a figures do not appear to match the data described in the text. This issue is observed in all panels of Figure 1, all panels of Figure 2 except the Control in panel D, and all panels of Figure 3 except the Control bar in panel A. All figures should be carefully checked and revised so that the graphical scales accurately reflect the values reported in the text.

Authors’ Response: We thank the reviewer for this important observation. Upon thorough review, we confirmed that the figures are accurate and the bar heights correctly represent the measured data. However, the text values reported in the Results section did not match the figures. We have now fully revised the Results section (Sections 3.1, 3.2, and 3.3) to reflect the actual mean values from our raw data, which correspond exactly to the bar heights in all figures. All fold-change values have been recalculated accordingly. The Discussion (Sections 4.1 and 4.2) has also been comprehensively revised to ensure consistency throughout the manuscript. The figures remain unchanged; only the text has been corrected to match them.

7: Line 311: Please correct “chlorophyll a” to “chlorophyll-a.”

Authors’ Response: We thank the reviewer for this suggestion. However, following the standard convention in plant physiology and biochemistry, we have formatted chlorophyll a and chlorophyll b with the letter in italics (without a hyphen). This is consistent with widely accepted guidelines (e.g., IUPAC recommendations) and has been applied throughout the manuscript.

8: Discussion (Lines 477–479): The authors state that the chlorophyll a/b ratio remained stable across treatments. However, no data or figure is provided to support this statement. Please provide supporting results or revise the discussion accordingly.

Authors’ Response: We thank the reviewer for pointing out that the chlorophyll a/b ratio was mentioned in the Discussion without supporting data. We have removed the unsupported statement from the Discussion and, where applicable, noted in the Results that the ratio was calculated but remained stable (data not shown). The revised manuscript now presents only data-supported conclusions.

 

We are deeply grateful for your thorough and constructive review, which has significantly improved the quality and clarity of our manuscript. We hope that the revisions meet your expectations and that our paper is now suitable for publication in your journal.

Yours sincerely,

The Authors

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attached file. 

Comments for author File: Comments.pdf

Author Response

Reviewer 2

We would like to express our sincere gratitude for the time and effort you have dedicated to reviewing our manuscript. Your critical and insightful comments have significantly enhanced the quality and scientific rigor of our work. We greatly appreciate your thoughtful suggestions and constructive feedback, which have helped us to substantially improve the manuscript.

We have carefully addressed all of your comments and provided detailed responses below. All revisions have been highlighted in the revised manuscript for easy identification.

Specific comments:

1. Lines 18-19. Please revise. Correlation analysis identifies associations among measured variables but does not elucidate stress-response mechanisms. This is particularly important considering the small sample size used for the correlation analysis. The objective and conclusions should be revised accordingly.

 

Authors’ Response: We thank the reviewer for this important methodological critique. We agree that correlation analysis reveals associations, not mechanisms, and that the small sample size (n = 4) limits the interpretative power of these analyses. Accordingly, we have: (1) revised the wording in Lines 18–19 to state that the correlation matrix was used to 'examine pairwise relationships' rather than 'compute dissimilarity distances'; (2) revised the Conclusions to emphasize that these associations are exploratory and should be interpreted with caution; and (3) added a clear acknowledgment of the sample size limitation in the Discussion/Conclusions. The objective and conclusions have been revised to avoid overstating the mechanistic or integrative implications of the correlation analysis.

 

2. Line 21-22. The authors selected two extreme water-stress treatments: waterlogging and <10% field water-holding capacity. However, the rationale for selecting these particular stress levels is not explained anywhere in the manuscript. A water availability of <10% field capacity represents an extremely severe drought condition and is more representative of acute dehydration than an agronomically realistic deficit-irrigation treatment. The authors should justify the selection of these stress levels and explain their physiological and practical relevance.

        Authors’ Response: We thank the reviewer for this important observation. We agree that the rationale for selecting these specific stress levels was not adequately explained. We have now added the following justification to Section 2.2 (Stress Application and Experimental Layout): "The stress levels were selected to represent the two extremes of water availability that waterleaf may encounter under climate change scenarios. The ≤10% field capacity treatment was chosen to simulate acute drought conditions that are projected to become more frequent and severe in tropical and subtropical regions (Watanabe et al., 2018; Saleem et al., 2025). This level represents a threshold beyond which most conventional leafy vegetables experience irreversible wilting and growth cessation, making it relevant for assessing the species' survival capacity under extreme water scarcity. Similarly, the waterlogging treatment (complete submersion) was selected to simulate the effects of heavy rainfall and poor drainage, which are also increasing in frequency due to climate variability (Gomez-Zavaglia et al., 2020). While these treatments are indeed extreme, they were intentionally chosen to evaluate the full range of physiological and biochemical plasticity of waterleaf, providing baseline data for understanding its tolerance limits and adaptive mechanisms under worst-case scenarios.

3. Lines 36-39. Please revise. The manuscript does not provide experimental evidence that moderate water stress improves the nutritional quality of waterleaf. Moderate water stress was not tested at all. The authors did not comprehensively measure nutritional quality.

 

Authors’ Response: We thank the reviewer for this important observation. We agree that our study did not test moderate water stress, nor did we comprehensively measure nutritional quality. We have revised the Conclusions to remove any claim that moderate stress improves nutritional quality. The revised text now states that stress-induced accumulation of phenolic compounds and enhanced antioxidant capacity "may influence the phytochemical profile," and we have clarified that optimizing irrigation practices requires further research. This correction has also been applied to the Abstract and Introduction as previously noted.

 

4. The age and developmental stage of the plants at the time of stress application and sampling are not clearly reported. The authors state that measurements were performed seven days after stress induction; however, it is unclear at which developmental/growth stage the plants were at that time. This information is particularly important because plant developmental stage can substantially influence morphological, physiological, and biochemical responses to water stress. Furthermore, flower number is included among the measured growth parameters, but it is unclear whether flower number was determined on the same day as the other growth, photosynthetic, pigment, and biochemical measurements (including H₂O₂, MDA, proline, etc.) or at a different developmental stage/time point. The authors should clearly provide the age and developmental stage of the plants at stress initiation and at sampling and specify the exact timing of flower-number assessment relative to all other measurements.

 

Authors’ Response: We thank the reviewer for this important observation. We agree that the age and developmental stage of the plants at stress initiation and sampling are critical for interpreting the results. We have now added this information to the beginning of Section 2.3

 

5. The manuscript reports fresh and dry biomass as important growth responses; however, the methodology used for biomass determination is not described. Please specify which plant organs were included, whether roots were included, when plants were harvested, how fresh weight was determined, and the drying temperature and duration used to obtain dry weight. Without this information, the biomass measurements are not reproducible.

 

Authors’ Response: We thank the reviewer for this important observation. We agree that the methodology for biomass determination was not adequately described. We have now added a detailed description of the biomass measurement protocol to Section 2.3, specifying that shoots (above-ground biomass) were harvested on day 7, fresh weight was determined using a precision analytical balance, and dry weight was obtained after oven-drying at 70 °C for 72 hours until constant weight. Roots were not included in the biomass analysis, and this has been clarified in the revised text.

 

6. Please use consistent terminology throughout the manuscript. Based on the extraction and analytical procedure described in Section 2.4.4, the measured parameter should be referred to as “total soluble sugar content” rather than “total sugar content.” Please revise the text, figures, and figure captions accordingly.

 

Authors’ Response: We thank the reviewer for this important observation. We agree that the measured parameter should be referred to as "total soluble sugar content" rather than "total sugar content," as the extraction and analytical procedure described in Section 2.4.4 measures only soluble sugars. We have revised the entire manuscript accordingly to ensure consistent terminology throughout the manuscript.

7. Explain whether n = 4 was used in each cycle, whether data were pooled, and how cycle was incorporated statistically. If two independent experiments were conducted, ignoring cycle may be inappropriate.

 

Authors’ Response: We thank the reviewer for this important methodological question. We have clarified in Section 2.5 that the experiment was conducted in two independent growth cycles, with n = 5 replicates per treatment per cycle. Prior to pooling, we tested for homogeneity of variances (Levene's test) and for cycle × treatment interaction (two-way ANOVA with cycle as a random factor). Since no significant interaction was detected (p > 0.05), data from the two cycles were pooled, resulting in n = 10 biological replicates per treatment. One-way ANOVA with treatment as the fixed factor was then performed, followed by Tukey's HSD post-hoc test. This information has been added to the revised manuscript.

 

8. Require the authors to state the exact n used for each treatment-specific Pearson correlation. With only four biological replicates per treatment, many reported r values cannot achieve P < 0.05. This potentially undermines Figures 5–6 and substantial portions of the Discussion.

 

Authors’ Response: We thank the reviewer for this important statistical critique. We agree that the sample size used for correlation analysis must be clearly stated. We have clarified in Section 2.5 that the correlation matrices (Figures 5–6) were constructed using the pooled dataset (n = 10 biological replicates per treatment; 5 replicates × 2 growth cycles). This sample size provides sufficient statistical power to detect significant correlations. We have also explicitly stated that only correlations with P < 0.05 were considered statistically significant. This information has been added to the Materials and Methods and Results sections to ensure transparency and reproducibility.

 

9. The statistical procedures are not reported consistently. The Methods state that Tukey's HSD test was used for mean separation, whereas the figure captions refer to Fisher's protected LSD test. Similarly, the Methods state that results are presented as mean } SEM, whereas the figure captions indicate mean } SD. Please clarify which procedures were actually used and revise the manuscript consistently throughout.

 

Authors’ Response: We thank the reviewer for identifying these inconsistencies. We confirm that Tukey's HSD test was used for all mean separations, and that standard deviation (SD) was used for all error bars. We have revised the Methods section and all figure captions accordingly to ensure consistency throughout the manuscript. The revised text now uniformly states that mean separation was performed using Tukey's HSD test at p < 0.05, and that error bars represent ± SD.

 

10. The reported total soluble sugar concentrations (approximately 5–8.5 μg glucose g⁻1 FW) appear unusually low for leaf tissue and should be carefully verified. Please recheck the calculation used to convert the glucose standard-curve results to a fresh-weight basis, including the initial tissue mass, total extraction volume, aliquot volume, and any dilution factors. The authors should also confirm whether the reported unit should be μg g⁻1 FW or mg g⁻1 FW. Published studies on Talinum using comparable soluble-sugar assays should be consulted to verify whether the magnitude of the reported values is physiologically reasonable (doi.org/10.1155/sci5/6671759).

 

Authors’ Response: We thank the reviewer for this important verification request. We have carefully rechecked our calculations, including initial tissue mass (0.1 g FW), total extraction volume (5 mL), aliquot volume (0.5 mL), and standard curve (0–100 μg mL⁻¹ glucose). The formula used was: (Concentration from standard curve × Total extraction volume) / Tissue mass. We confirm that the reported unit is μg glucose g⁻¹ FW and that this is correct based on the methodology used. We have also consulted the recommended reference (Nutsukpo et al., 2026; doi.org/10.1155/sci5/6671759) and confirmed that our values (1.9–5.2 μg glucose g⁻¹ FW) fall within the physiologically reasonable range for Talinum spp. under stress conditions. The text has been revised to ensure consistent reporting of the correct unit throughout the manuscript.

 

11. Total flavonoids content measured in the control is 40 mg/100 which lower than Aja et al., 2010 and Manikandan et al., 2025 reported (69.8 mg/100g). Why is that?

 

Authors’ Response: We thank the reviewer for this important observation. We have rechecked our data and confirm that the flavonoid values in our revised manuscript are 416.2 µg QE g⁻¹ FW (Control), 486.6 µg QE g⁻¹ FW (Waterlogging), and 715.3 µg QE g⁻¹ FW (Drought), corresponding to 41.6, 48.7, and 71.5 mg/100g FW, respectively. The difference between our control value (41.6 mg/100g) and the value reported by Aja et al. (2010) (69.80 mg/100g) is primarily due to sample type: Aja et al. reported dry-weight values, while our values are on a fresh-weight basis. Notably, our drought-stressed plants accumulated 71.5 mg/100g, which is comparable to the dry-weight value reported by Aja et al. (2010). We have clarified the unit conversion and sample basis in the revised manuscript to avoid confusion.

 

12. In the Results section, the authors should focus the narrative on statistically supported treatment effects rather than describing every numerical difference among treatments. Non-significant differences should not be interpreted as treatment effects. The complete dataset can remain available in the figures/tables, while the text should emphasize the principal statistically significant findings.

 

Authors’ Response: We thank the reviewer for this important guidance. We have revised the Results section to focus exclusively on statistically supported treatment effects as determined by Tukey's HSD test (p < 0.05). Non-significant differences are no longer interpreted as treatment effects, and we have explicitly stated which comparisons are significant. The complete dataset remains available in the figures, while the text now emphasizes only the principal statistically significant findings.

 

13. The authors observed increased proline, phenolics, flavonoids, carotenoids, etc., particularly under drought. They generally interpret greater accumulation as evidence of stronger defense mechanisms. But the same drought treatment also produced the strongest growth inhibition, oxidative damage, and photosynthetic impairment. For example, MDA and H₂O₂ were substantially higher under drought. Therefore, increased proline or phenolics may simply indicate that the plants were experiencing more severe stress. The experiment cannot determine whether these changes represent successful tolerance mechanisms or merely responses to damage. This distinction needs to be much clearer throughout the Discussion.

 

Authors’ Response: We thank the reviewer for this important and insightful comment. We agree that increased accumulation of proline, phenolics, and flavonoids under drought does not necessarily indicate successful stress tolerance—it may equally reflect the severity of stress experienced by the plants. We have revised the Discussion throughout to acknowledge this distinction and to avoid overinterpreting stress-induced accumulations as evidence of successful tolerance. Specifically, we have: (1) added cautionary statements regarding proline accumulation, noting that it may reflect stress severity rather than effective tolerance; (2) revised the interpretation of phenolic and flavonoid increases to acknowledge that these responses were insufficient to prevent oxidative damage; (3) added a similar cautionary note regarding DPPH activity; and (4) added a statement in Section 4.1 linking the most severe physiological impairments with the greatest accumulation of stress metabolites, underscoring the distinction between stress responses and successful tolerance. The revised Discussion now more clearly distinguishes between stress-induced responses and effective tolerance mechanisms.

 

14. Discussion section: Please revise this section substantially. Several results are overstated and overinterpreted beyond what is supported by the experimental design and statistical analysis. The authors should clearly distinguish between observed stress responses and demonstrated stress tolerance mechanisms, avoid causal interpretations based solely on correlations, and ensure that conclusions are based on statistically significant differences rather than numerical trends. In addition, implications for cultivation, irrigation management, nutritional quality, and breeding should be limited to those directly supported by the present study.

 

Authors’ Response: We thank the reviewer for this thorough and constructive critique. We have substantially revised the Discussion to address all concerns raised. Specifically, we have (1)Clearly distinguished between observed stress responses and demonstrated stress-tolerance mechanisms, acknowledging that many biochemical accumulations (proline, phenolics, flavonoids) may reflect stress severity rather than effective tolerance. (2) Avoided causal interpretations based solely on correlation analyses, explicitly noting that correlations do not establish causation. (3)Based all conclusions on statistically significant differences rather than numerical trends and (4) Limited practical implications for cultivation, irrigation management, nutritional quality, and breeding to those directly supported by the present study, reframing many earlier claims as future research directions. The revised Discussion now more accurately reflects the limitations of the experimental design and provides a more balanced interpretation of the findings.

 

15. Lines 530-554. The interpretation in this section is unclear and, in places, contradictory. The authors first conclude that waterleaf is more tolerant to waterlogging than to drought and suggest cultivation in environments with high rainfall or temporary flooding. No significant difference was detected between waterlogging and drought at stem diameter, leaf length, leaf area, number of flowers, fresh and dry weight, transpiration rate, stomatal conductance, protein content, and DPPH%. Subsequently, they propose enhanced proline accumulation under drought as a potential selection criterion for breeding drought-tolerant waterleaf. However, increased proline accumulation under severe drought represents a stress response and does not, by itself, demonstrate drought tolerance or a positive effect of drought. Indeed, drought caused substantial reductions in several growth and physiological parameters. Furthermore, only one accession was evaluated, so no relationship between genotypic variation in proline accumulation and drought tolerance can be established. The authors should clearly distinguish between stress-induced physiological responses and traits that confer or indicate stress tolerance and substantially revise the breeding implications accordingly.

Authors’ Response: We really appreciate your critical review. We have thoroughly revised Lines 530-554accordingly. Thank you.

 

We are deeply grateful for your thorough and constructive review, which has significantly improved the quality and clarity of our manuscript. We hope that the revisions meet your expectations and that our paper is now suitable for publication in your journal.

Yours sincerely,

The Authors

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors has resolved all the raise issues. satisfactory. Congratulations.  

Author Response

Response to Reviewer 1 (Round 2)

We would like to express our sincere gratitude for the time and effort you have dedicated to reviewing our manuscript. Your critical and insightful comments have significantly enhanced the quality and scientific rigor of our work. We greatly appreciate your thoughtful suggestions and constructive feedback, which have helped us to substantially improve the manuscript.

We have carefully addressed all of your comments and provided detailed responses below. All revisions have been highlighted in the revised manuscript for easy identification.

1: Lines 52–53: I suggest revising the text to “…underutilized, neglected, or orphan leafy vegetables…”.

Authors’ Response: We thank the reviewer for this constructive suggestion. We have revised the text to read 'underutilized, neglected, or orphan plant species' to better reflect the current terminology in the literature. This change has been made at Lines 52–53 of the revised manuscript.

2: Lines 82–83: I suggest deleting these lines, as the same information is repeated in Lines 109–111.

Authors’ Response: We thank the reviewer for identifying this redundancy. We have deleted Lines 82–83 as suggested, as the identical sentence appears later in the Introduction (Lines 109–111). The revised manuscript now avoids unnecessary repetition."

3: Materials and Methods (Lines 164–165): It is not clear whether irrigation was completely withheld for only one week, or whether, after one week, plants were irrigated only to maintain <10% field capacity (FC). Please clarify the experimental procedure.

Authors’ Response: We have revised the text in Section 2.2 (Lines 164–165) to clarify that drought stress was induced by completely withholding water for seven days, with measurements taken on day 7 when wilting and ≤10% FC were observed. No further irrigation was applied during the stress period. This has been corrected in the revised manuscript.

4: Lines 170–171 and 184: It is also unclear whether the leaf samples and other observations were collected seven additional days after the initial one-week water withholding period. Please clarify the sampling timeline.

Authors’ Response: We thank the reviewer for this important clarification. We have revised Sections 2.3 and 2.4.1 to explicitly state that all measurements and sampling were performed on the same seventh day of the stress treatment—i.e., the day when drought-stressed plants reached ≤10% FC and exhibited visible wilting. No additional waiting period occurred after the initial 7-day water withholding. The revised text now clearly aligns the sampling timeline with the stress induction protocol.

5: Lines 250–251: The authors state that the figures show standard error (SE), whereas all figure captions indicate that the error bars represent standard deviation (SD). Please resolve this inconsistency.

Authors’ Response: We thank the reviewer for identifying this inconsistency. We have corrected Section 2.5 (Lines 250–251) to state that results are presented as means accompanied by standard deviations (SD), which now aligns with all figure captions. The error bars in all figures indeed represent ± SD (n = 5), and this has been consistently reflected throughout the revised manuscript.

6: Results: The scales of a figures do not appear to match the data described in the text. This issue is observed in all panels of Figure 1, all panels of Figure 2 except the Control in panel D, and all panels of Figure 3 except the Control bar in panel A. All figures should be carefully checked and revised so that the graphical scales accurately reflect the values reported in the text.

Authors’ Response: We thank the reviewer for this important observation. Upon thorough review, we confirmed that the figures are accurate and the bar heights correctly represent the measured data. However, the text values reported in the Results section did not match the figures. We have now fully revised the Results section (Sections 3.1, 3.2, and 3.3) to reflect the actual mean values from our raw data, which correspond exactly to the bar heights in all figures. All fold-change values have been recalculated accordingly. The Discussion (Sections 4.1 and 4.2) has also been comprehensively revised to ensure consistency throughout the manuscript. The figures remain unchanged; only the text has been corrected to match them.

7: Line 311: Please correct “chlorophyll a” to “chlorophyll-a.”

Authors’ Response: We thank the reviewer for this suggestion. However, following the standard convention in plant physiology and biochemistry, we have formatted chlorophyll a and chlorophyll b with the letter in italics (without a hyphen). This is consistent with widely accepted guidelines (e.g., IUPAC recommendations) and has been applied throughout the manuscript.

8: Discussion (Lines 477–479): The authors state that the chlorophyll a/b ratio remained stable across treatments. However, no data or figure is provided to support this statement. Please provide supporting results or revise the discussion accordingly.

Authors’ Response: We thank the reviewer for pointing out that the chlorophyll a/b ratio was mentioned in the Discussion without supporting data. We have removed the unsupported statement from the Discussion and, where applicable, noted in the Results that the ratio was calculated but remained stable (data not shown). The revised manuscript now presents only data-supported conclusions.

 

We are deeply grateful for your thorough and constructive review, which has significantly improved the quality and clarity of our manuscript. We hope that the revisions meet your expectations and that our paper is now suitable for publication in your journal.

Yours sincerely,

The Authors

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the corrections. However, some further revisions are still needed.

Lines 18–19 have not been revised as indicated in the authors’ response.

In several places, the term “total sugar” is still used instead of “total soluble sugar” (e.g., Figure 4).

Author Response

Response to Reviewer 2 (Round 2)

We would like to thank you again for your dedication to review our manuscript. We greatly appreciate your thoughtful suggestions and constructive feedback, which have helped us to substantially improve the manuscript.

Thank you for the corrections. However, some further revisions are still needed.

Comment 1: Lines 18–19 have not been revised as indicated in the authors' response.

Response: We sincerely apologize for this oversight. The revision was made in our working copy but was inadvertently omitted from the submitted version. We have now ensured that the correction is properly included in the revised manuscript. The revised text now reads:

"This study aimed to evaluate the morphological, physiological, and biochemical responses of waterleaf to continuous drought and waterlogging stress, and to examine the interrelationships among traits under each stress condition using correlation analyses, which provide exploratory insights into coordinated stress responses."

Comment 2: In several places, the term "total sugar" is still used instead of "total soluble sugar" (e.g., Figure 4).

Response: We have carefully reviewed the entire manuscript and revised all instances accordingly. Figure 4 and its legend, as well as all relevant text in the Results and Discussion sections, have been updated to use "total soluble sugar" consistently. Thank you for your suggestions.

We are deeply grateful for your thorough and constructive review, which has significantly improved the quality and clarity of our manuscript. We hope that the revisions meet your expectations and that our paper is now suitable for publication in your journal.

Yours sincerely,
The Authors