Terminalia arjuna Switches from Adaptive to Survival Strategy Under Severe Water Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Drought causes osmotic stress, which is accompanied by disruption of cellular metabolism and the development of oxidative stress, which ultimately has a detrimental effect on plant productivity. Therefore, it is important to develop and implement sustainable agricultural production methods that will reduce the negative impacts of abiotic stressors and ensure future food security.

One possible approach is to study the morphophysiological and biochemical changes in plant seedlings depending on the level of water stress.

The study was conducted on Terminalia arjuna (Arjun), a tropical deciduous tree with a valuable medicinal value. A large number of morphological, biochemical, and physiological parameters were assessed, along with changes in nutrient absorption, and adaptation strategies were identified depending on the intensity of water stress (control (100% F_wc), mild-stress (75% F_wc), moderate-stress (50% F_wc), and severe-stress (25% F_wc). The growth parameters of the seedlings, stomatal density, stomatal conductance, transpiration rate, photosynthesis rate, water use efficiency, chlorophyll and carotenoid content, membrane stability index, relative water content, proline content, malondialdehyde, hydrogen peroxide, leaf mineral contents, and a number of other indicators were determined.

Thus, in the course of their study, the authors of the article identified the mechanisms by which T. arjuna adapts to moderate water stress (50% of field capacity) and switches to survival-oriented strategies under severe stress (25% of field capacity).

The article corresponds to the profile of the journal.

The experiments were conducted using classical analytical methods, and the obtained data were statistically processed. An analysis of variance was performed for all parameters.

The work is well illustrated.

The study is relevant, as the effects of abiotic factors can have serious consequences for food security and the economies of many countries.

Among the limitations of the study are the following:

• The content of physiological parameters (proline, MDA) was calculated on a fresh weight basis (not on a dry weight basis), which is incorrect, as the study discusses the effects of drought on plants.
• The MDA determination method used by the authors is not specific enough to be considered a determination of MDA. It only detects TBARS (thiobarbituric acid reactive substances). Even the use of 1-methyl-2-phenylindole, which increases the specificity of the reaction in animal tissues, is not very suitable for plants, mainly due to the phenolic and carbohydrate compounds. Thus, HPLC is recommended as the most robust method for measuring MDA and HNE in plant tissues. In all other cases, it is recommended to talk about the determination of TBARS, not MDA (Johnston et al. 2007. Plant Physiol. Bioch. 45, 108–112. DOI: 10.1016/j.plaphy.2007.01.011).
• The bibliography is poorly formatted: (a) The article title is sometimes capitalized, sometimes lowercase – references 15, 35, 36, 38, 40, etc.; (b) the Latin names of plants are not italicized – references 14, 36, and etc.

I believe the study can be published after revision.

Author Response

1. The content of physiological parameters (proline, MDA) was calculated on a fresh weight basis (not on a dry weight basis), which is incorrect, as the study discusses the effects of drought on plants.

Authors response:

Thank you for pointing this out. Following the reviewer’s suggestion, we reanalyzed the biochemical parameters using dry weight instead of fresh weight. The stored leaf samples were used to determine the proline, TBARS and H₂O₂ contents on a dry mass basis to ensure greater accuracy, particularly under drought conditions. The revised results are now demonstrated in the final manuscript.

2.The MDA determination method used by the authors is not specific enough to be considered a determination of MDA. It only detects TBARS (thiobarbituric acid reactive substances). Even the use of 1-methyl-2-phenylindole, which increases the specificity of the reaction in animal tissues, is not very suitable for plants, mainly due to the phenolic and carbohydrate compounds. Thus, HPLC is recommended as the most robust method for measuring MDA and HNE in plant tissues. In all other cases, it is recommended to talk about the determination of TBARS, not MDA (Johnston et al. 2007. Plant Physiol. Bioch. 45, 108–112. DOI: 10.1016/j.plaphy.2007.01.011).

Authors response:

Thank you for pointing out this important issue and for the helpful reference. I agree that the TBA assay used in this study measures TBARS rather than MDA. Following the reviewer’s suggestion, I have revised the manuscript and replaced the term MDA with TBARS throughout the Methodology, Results, and Discussion sections to ensure accurate representation of the analytical method. The recommended reference has also been considered and cited in the revised manuscript.

3.The bibliography is poorly formatted: (a) The article title is sometimes capitalized, sometimes lowercase – references 15, 35, 36, 38, 40, etc.; (b) the Latin names of plants are not italicized – references 14, 36, and etc.

Authors response:

I carefully reviewed the entire reference list and corrected all the formatting inconsistencies.

Reviewer 2 Report

Comments and Suggestions for Authors

I have read the manuscript Plants - 4178508. First, I would like to thank you for presenting your results. The title „Terminalia arjuna Switches from Adaptive to Survival Strategy under Severe Water Stress” written by Lumat Afrin Jui et al., is sugestive and siutable for the content of this work.

The abstract is well structured and includes clear quantitative data that support the conclusions. I recommend completing it with a sentence that explicitly highlights the original contribution of the study, namely the simultaneous integration of morphological, physiological, biochemical and mineral redistribution parameters in T. arjuna under gradual water stress.

The introduction provides a solid context regarding the importance of the species and the impact of drought in a climatic context, and the literature is up-to-date and relevant. As a recommendation, a clearer statement of the hypotheses regarding the differentiation of the response to moderate versus severe stress and the role of ionic redistribution in stomatal regulation and water use efficiency. I also recommend replacing the word “crucial” with another synonym, it appears in several sections in your paper, it is a word used by artificial intelligence.

The materials and methods are described in detail and include validated standard techniques (porometry, photosynthesis, ICP-OES, Kjeldahl method), which confers experimental credibility. However, the number of replicates is low, and in the ANOVA tables the factor is called “Tree_ID” instead of “Treatment”, which may create methodological confusion. The experimental unit is not clearly defined and should be explicitly stated. Also, identical repetitions of ANOVA values ​​occur for certain parameters, suggesting a possible reporting error that should be checked before publication.

The statistical analysis is correctly applied from the perspective of univariate tests (ANOVA and Tukey), but remains limited to separate comparisons for each parameter. Given the complexity of the data set, it would be advisable to include a multivariate analysis or a correlation matrix to more robustly support the idea of ​​a transition from adaptation to survival strategy.

The morphological results are coherent and well supported graphically, and the increase in root biomass under moderate stress is an interesting aspect. However, the root:shoot ratio is not presented explicitly, although it would directly support the adaptive interpretation.

The physiological results clearly show a reduction in stomatal conductance and photosynthesis, and the increase in WUE is well documented. The interpretation of the increase in WUE is cautiously formulated in the discussion, but it should be emphasized more clearly that this increase reflects a functional trade-off, not an improvement in performance.

The biochemical results are consistent with the literature, demonstrating proline accumulation and increased oxidative stress markers. However, the lack of assessment of antioxidant enzymes limits the mechanistic interpretation of cellular protection.

The nutrient results represent one of the valuable contributions of the study, especially highlighting the increased accumulation of K and Ca under stress.

The discussion is extensive and well connected to recent literature, but contains some repetitions and interpretative formulations. It would benefit from a clearer structuring around the hypotheses and the conceptual integration of the responses into a synthetic model.

The conclusions adequately summarize the results and highlight practical implications, but should place a clearer emphasis on the scientific contribution, not just on applications in nurseries or afforestation programs.

The supplementary material contains the detailed ANOVA analysis, but presents numerical inconsistencies and improper naming of factors, which should be corrected to ensure statistical rigor.

Final verdict: The study has good scientific value and is publishable after minor revisions to clarify the statistical methodology, explicitly formulate the hypotheses, correct the ANOVA tables, and reduce speculative formulations regarding physiological mechanisms.

I recommend publication after minor revisions.

Congratulations to the authors!

Author Response

1.The abstract is well structured and includes clear quantitative data that support the conclusions. I recommend completing it with a sentence that explicitly highlights the original contribution of the study, namely the simultaneous integration of morphological, physiological, biochemical and mineral redistribution parameters in T. arjuna under gradual water stress.

Authors response:

Thank you for your positive evaluation and suggestion. I tried to added a sentence which highlights the original contribution of the study, emphasizing the simultaneous integration of all parameters to assess the response of T. arjuna with increasing stress severity. This addition better clarifies the novelty and scientific aspect of the study.

2.The introduction provides a solid context regarding the importance of the species and the impact of drought in a climatic context, and the literature is up-to-date and relevant. As a recommendation, a clearer statement of the hypotheses regarding the differentiation of the response to moderate versus severe stress and the role of ionic redistribution in stomatal regulation and water use efficiency. I also recommend replacing the word “crucial” with another synonym, it appears in several sections in your paper, it is a word used by artificial intelligence.

Authors response:

Following your recommendation, I tried to illustrate the research hypotheses in the introduction section, particularly emphasizing the expected differentiation between moderate and severe water stress responses, as well as the proposed role of ionic redistribution in stomatal regulation and water use efficiency. In addition, we have carefully revised the manuscript and replaced the word “crucial” with more appropriate scientific synonyms.

3.The materials and methods are described in detail and include validated standard techniques (porometry, photosynthesis, ICP-OES, Kjeldahl method), which confers experimental credibility. However, the number of replicates is low, and in the ANOVA tables the factor is called “Tree_ID” instead of “Treatment”, which may create methodological confusion. The experimental unit is not clearly defined and should be explicitly stated. Also, identical repetitions of ANOVA values ​​occur for certain parameters, suggesting a possible reporting error that should be checked before publication.

Authors response:

Thank you for giving positive view. Yes, I agree with you, the number of replications are low, but we had some experimental constraints using a higher number of replicate seedlings, however, I have clearly mentioned the experimental unit in the revised manuscript. In addition, I have corrected the terminology in the ANOVA tables by replacing “Tree_ID” with “Treatment”. I have also carefully rechecked and corrected all ANOVA outputs and statistical tables to verify the reported values.

 

4.The statistical analysis is correctly applied from the perspective of univariate tests (ANOVA and Tukey), but remains limited to separate comparisons for each parameter. Given the complexity of the data set, it would be advisable to include a multivariate analysis or a correlation matrix to more robustly support the idea of ​​a transition from adaptation to survival strategy.

Authors response:

I completely agree with you. In response, I have included Pearson correlation analysis in the supplementary data and a heatmap cluster in the main manuscript to better illustrate the relationships among all the parameters.

5.The morphological results are coherent and well supported graphically, and the increase in root biomass under moderate stress is an interesting aspect. However, the root: shoot ratio is not presented explicitly, although it would directly support the adaptive interpretation.

Authors response:

I have now added the root: shoot ratio finding in the result and discussion section. The plot for root: shoot ratio was added in the supplementary data.

 

6.The physiological results clearly show a reduction in stomatal conductance and photosynthesis, and the increase in WUE is well documented. The interpretation of the increase in WUE is cautiously formulated in the discussion, but it should be emphasized more clearly that this increase reflects a functional trade-off, not an improvement in performance.

Authors response:

Thank you very much for your insightful comment. I have tried to rewrite the discussion part for increased WUE better portraying the functional trade of adaptive phenomenon.

7.The biochemical results are consistent with the literature, demonstrating proline accumulation and increased oxidative stress markers. However, the lack of assessment of antioxidant enzymes limits the mechanistic interpretation of cellular protection.

Authors response:

I completely agree with your observation, but due to some technical and logistical constraints during the experimental period, it was not possible to assess antioxidant enzyme activities in this study. I mentioned the limitation of our study in the conclusion.

8.The nutrient results represent one of the valuable contributions of the study, especially highlighting the increased accumulation of K and Ca under stress.

Authors response:

Thank you very much for your positive evaluation and recognition of this aspect of our study.

9.The discussion is extensive and well connected to recent literature, but contains some repetitions and interpretative formulations. It would benefit from a clearer structuring around the hypotheses and the conceptual integration of the responses into a synthetic model.

Authors response:

Thank you for positive assessment of the discussion section. Following your suggestion, I have carefully revised and restructured the discussion to reduce repetitions and interpretative structure, and tried to improve the logical flow.

10.The conclusions adequately summarize the results and highlight practical implications, but should place a clearer emphasis on the scientific contribution, not just on applications in nurseries or afforestation programs.

Authors response:

I have revised the conclusion section and added the scientific contribution and theoretical implications of the study along with its practical applications.

11.The supplementary material contains the detailed ANOVA analysis, but presents numerical inconsistencies and improper naming of factors, which should be corrected to ensure statistical rigor.

Authors response:

Thank you for pointing out these issues in the supplementary material. I have carefully reviewed the detailed ANOVA tables and corrected the identified numerical inconsistencies and improper naming of statistical factors to ensure accuracy and consistency.

12.Final verdict: The study has good scientific value and is publishable after minor revisions to clarify the statistical methodology, explicitly formulate the hypotheses, correct the ANOVA tables, and reduce speculative formulations regarding physiological mechanisms.

Authors response:

I sincerely thank the reviewer for the overall positive evaluation and constructive feedback. I appreciate the recognition of the scientific value of our study. In accordance with the reviewer’s recommendations, I have carefully revised the manuscript to clarify the statistical methodology, illustrating the research hypotheses, correct and standardize the ANOVA tables, and reduce speculative interpretations. All revisions have been implemented to improve the scientific strength of the manuscript. I believe these changes have significantly improved the quality of the paper.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Relevant experimental details need to be supplemented. For example, the number of rinses with sterile water after the 70% ethanol treatment should be specified to avoid the impact of disinfectant residue on germination. Additionally, the reason for not measuring stomatal aperture/pore size should be explained. The specific method for calculating stomatal density using ImageJ and the number of fields of view should also be supplemented. In the "2.1. Experimental design" section, the author mentions that the experiment was conducted from "October 2023 to March 2023." This is chronologically impossible. Please verify and correct the specific years.
2. The overall image quality of the article needs improvement. For example, the experimental flow chart in Figure 1 appears somewhat informal, and the clarity of some icons is insufficient. It is recommended to redraw the flow chart using more professional scientific illustration tools to enhance the overall academic quality of the article.The scale bar in the plant morphology photos of Figure 3 is blurry, so a clear scale bar needs to be added. For the stomatal micrographs in Figure 5, complete images of the 4 treatments need to be supplemented, which can be added to the supplementary figures. The method clearly states that the experiment was divided into 4 treatment groups, with 3 replicates per group, resulting in a total sample size of 12. However, in the captions of each figure (such as Figure 2, 4, 6, 7, 8), it is marked as "(n=12)". The "n" in a figure caption usually refers to the number of biological replicates per treatment group. Writing "(n=12)" would lead readers to mistakenly believe that each bar represents the mean of 12 samples. It is recommended to correct this to "(n=3)".
3. The full text only performs inter-group difference analysis on single indicators, without conducting multivariate statistical analyses such as correlation analysis or principal component analysis (PCA). As a result, the synergistic/antagonistic relationships among various physiological and biochemical indicators cannot be clarified. For example, the correlation between the decline in photosynthetic rate and chlorophyll content, stomatal conductance, and leaf N/Mg content; the correlation between the increase in WUE and K/Ca accumulation; and the correlation between proline accumulation and membrane stability and oxidative damage are all core evidence for verifying the drought response mechanism. Some combined statistical analyses need to be supplemented to improve the logical coherence of the results.
4. The article discusses the increased accumulation of potassium and calcium under severe water stress and points out that this is related to stomatal regulation and the maintenance of carbon dioxide fixation. It is recommended to further strengthen the physiological mechanism network of how Ca ions, as signaling molecules, specifically induce stomatal closure in the discussion section. This would make the article's conclusion (the shift from adaptation to survival strategy) more compelling.The article found that under mild and moderate water stress, the root dry matter mass not only did not decrease but actually increased. In the "Discussion" section, the author primarily explains this from the perspective of nutrient uptake (e.g., nitrogen). To elevate the level of the discussion, it is recommended that the author incorporate a discussion on the potential molecular regulatory networks underlying root system architecture and adaptive growth. Furthermore, under moderate and severe stress, the plant's photosynthetic rate and transpiration rate decreased significantly. The author attributes this to an increase in water use efficiency (WUE) and adaptation as a survival strategy. It is suggested that the discussion also address whether the decline in these gas exchange parameters has a direct causal relationship with leaf senescence, abscission (the article mentions observing leaf browning and abscission after 30 days of severe stress), and the degradation of photosynthetic pigments (chlorophyll decreased by about one-third to one-half), rather than being solely a result of stomatal regulation.
5. There are multiple spelling and formatting errors in the article. It is recommended to revise and carefully proofread it. For example, in line 18, "constrain" should be the noun "constraint".In the flowchart of Figure 1, "Electron microschope" should be corrected to "Electron microscope".The citation "Staniak et al., 2024" mentioned in the discussion is not listed in the references section and needs to be added.

Comments on the Quality of English Language

Need to carefully check for spelling, grammar, and formatting issues.

Author Response

1.Relevant experimental details need to be supplemented. For example, the number of rinses with sterile water after the 70% ethanol treatment should be specified to avoid the impact of disinfectant residue on germination. Additionally, the reason for not measuring stomatal aperture/pore size should be explained. The specific method for calculating stomatal density using ImageJ and the number of fields of view should also be supplemented. In the "2.1. Experimental design" section, the author mentions that the experiment was conducted from "October 2023 to March 2023." This is chronologically impossible. Please verify and correct the specific years.

Authors response:

Thank you for your meaningful comments. I have carefully revised the manuscript to address these concerns. The experimental details have been clarified and added in the Methodology section, including the number of rinses with sterile water after the 70% ethanol treatment. In addition, the procedure for calculating stomatal density using ImageJ has been clearly described, including the number of microscopic fields of view analyzed. Following the reviewer’s suggestion, we have also included the measurement of stomatal size and added the methodological description in the Methodology section. Furthermore, the chronological error in Section 2.1 (Experimental Design) has been corrected which was a typing error, and the experimental period has been revised to reflect the correct timeline in the manuscript.

2.The overall image quality of the article needs improvement. For example, the experimental flow chart in Figure 1 appears somewhat informal, and the clarity of some icons is insufficient. It is recommended to redraw the flow chart using more professional scientific illustration tools to enhance the overall academic quality of the article. The scale bar in the plant morphology photos of Figure 3 is blurry, so a clear scale bar needs to be added. For the stomatal micrographs in Figure 5, complete images of the 4 treatments need to be supplemented, which can be added to the supplementary figures. The method clearly states that the experiment was divided into 4 treatment groups, with 3 replicates per group, resulting in a total sample size of 12. However, in the captions of each figure (such as Figure 2, 4, 6, 7, 8), it is marked as "(n=12)". The "n" in a figure caption usually refers to the number of biological replicates per treatment group. Writing "(n=12)" would lead readers to mistakenly believe that each bar represents the mean of 12 samples. It is recommended to correct this to "(n=3)".

Authors response:

Thank you for suggestions. We have rerun and reinserted all the figures to improve their overall quality and clarity. The experimental flow chart in Figure 1 has been redrawn using a more professional format to enhance its scientific presentation. The scale bar in the plant morphology images in Figure 3 has also been replaced with a clearer version. In addition, complete stomatal micrographs representing all four treatments have been provided and included as supplementary figure 4 to improve the completeness of the dataset. Furthermore, we have corrected the sample size notation (n=3) in the figure captions.

3.The full text only performs inter-group difference analysis on single indicators, without conducting multivariate statistical analyses such as correlation analysis or principal component analysis (PCA). As a result, the synergistic/antagonistic relationships among various physiological and biochemical indicators cannot be clarified. For example, the correlation between the decline in photosynthetic rate and chlorophyll content, stomatal conductance, and leaf N/Mg content; the correlation between the increase in WUE and K/Ca accumulation; and the correlation between proline accumulation and membrane stability and oxidative damage are all core evidence for verifying the drought response mechanism. Some combined statistical analyses need to be supplemented to improve the logical coherence of the results.

Authors response:

I completely agree with you. To better explore the relationships among the physiological and biochemical parameters, we have performed Pearson correlation analysis among the measured variables. The results are presented as a heatmap, which has been added to the main manuscript to illustrate the relationships among the indicators. In addition, a correlation matrix plot has been provided as supplementary material to further support the interpretation of these relationships. These additional analyses help clarify the associations among photosynthetic traits, nutrient accumulation, osmolyte production, and oxidative stress indicators, thereby improving the overall logical coherence of the results and discussion.

4.The article discusses the increased accumulation of potassium and calcium under severe water stress and points out that this is related to stomatal regulation and the maintenance of carbon dioxide fixation. It is recommended to further strengthen the physiological mechanism network of how Ca ions, as signaling molecules, specifically induce stomatal closure in the discussion section. This would make the article's conclusion (the shift from adaptation to survival strategy) more compelling. The article found that under mild and moderate water stress, the root dry matter mass not only did not decrease but actually increased. In the "Discussion" section, the author primarily explains this from the perspective of nutrient uptake (e.g., nitrogen). To elevate the level of the discussion, it is recommended that the author incorporate a discussion on the potential molecular regulatory networks underlying root system architecture and adaptive growth.

Authors response:

Thank you for this insightful suggestion. I have rewritten the specific discussion section elaborating the role of Ca²⁺ as a signaling molecule in stomatal regulation, and expanded the explanation of how Ca²⁺ signaling pathways contribute to stomatal closure under severe water stress. In addition, I have improved the discussion on the observed increase in root dry matter under mild and moderate stress. Beyond nutrient uptake explanations, I included a more detailed interpretation of the potential molecular regulatory networks governing root system architecture and adaptive growth responses, integrating hormonal regulation and stress-responsive signaling pathways.

5.Furthermore, under moderate and severe stress, the plant's photosynthetic rate and transpiration rate decreased significantly. The author attributes this to an increase in water use efficiency (WUE) and adaptation as a survival strategy. It is suggested that the discussion also address whether the decline in these gas exchange parameters has a direct causal relationship with leaf senescence, abscission (the article mentions observing leaf browning and abscission after 30 days of severe stress), and the degradation of photosynthetic pigments (chlorophyll decreased by about one-third to one-half), rather than being solely a result of stomatal regulation.

Authors response:

I have revised the discussion section and added the potential direct relationship between prolonged water stress, leaf senescence, abscission, and the decrease in photosynthetic efficiency, which were observed in the form of leaf browning and shedding after 30 days of severe stress. This interpretation strengthens the causal relationship between physiological decline and morphological deterioration, rather than explaining the results through only stomatal limitation.

6.There are multiple spelling and formatting errors in the article. It is recommended to revise and carefully proofread it. For example, in line 18, "constrain" should be the noun "constraint". In the flowchart of Figure 1, "Electron microschope" should be corrected to "Electron microscope". The citation "Staniak et al., 2024" mentioned in the discussion is not listed in the references section and needs to be added.

Authors response:

Thank you for carefully reviewing the manuscript and pointing out these errors. I have thoroughly read the manuscript and corrected all identified spelling and formatting mistakes, including changing “constrain” to “constraint” and correcting “Electron microschope” to “Electron microscope” in Figure 1. In addition, the missing citation “Staniak et al., 2024” has now been properly included in the reference list and cited accordingly in the manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Please make sure the font formatting for numbers and letters is uniform in all images.

Author Response

Reviewer Comment:
Please make sure the font formatting for numbers and letters is uniform in all images.

Response:
Thank you for this valuable comment. The font formatting for numbers and letters has been carefully checked and standardized across all figures to ensure consistency throughout the manuscript. The revised figures have been updated accordingly in the manuscript.