Review Reports - Sinorhizobium meliloti GAU-93 Inoculation Is Associated with Reduced Drought-Induced Oxidative Damage and Enhanced Flavonoid Accumulation and Antioxidant Capacity in Alfalfa

Round 1

Reviewer 1 Report (Previous Reviewer 1)

For this reason, the manuscript is now suitable for publication in Antioxidants in its current form.

Author Response

Comments 1:

After carefully reviewing the modified manuscript and the corrections made by the authors, I believe that the authors have made the requested corrections; thus, the manuscript has improved considerably. For this reason, the manuscript is now suitable for publication in Antioxidants in its current form.

Response: We sincerely thank the reviewer for the positive evaluation of our revised manuscript. We greatly appreciate the reviewer’s careful assessment and constructive comments throughout the review process, which have substantially improved the quality and clarity of the manuscript. We are pleased that the revised manuscript is now considered suitable for publication in Antioxidants.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 2)

General comments: I appreciate the considerable effort made by the authors in revising the manuscript. The revised version successfully addresses the vast majority of the concerns raised during the previous review.

The experimental design and statistical analysis have been substantially strengthened through the adoption of an appropriate two-way ANOVA,
the M&M section now provides sufficient methodological detail,
the Results have been revised to ensure consistency with both the statistical analyses and the figures.
The graphical presentation has also been improved, making the data easier to interpret.
The Discussion and Conclusions now adopt a more cautious interpretation of the findings and avoid unsupported mechanistic claims.

Overall, the manuscript has improved considerably and is suitable for publication after minor revision.

Specific comments:

Title: could be slightly moderated to better reflect the associative nature of the evidence presented.

The expression "by promoting" implies a causal mechanism that was not directly demonstrated in the present study. A title based on "is associated with" or equivalent wording would be more consistent with the cautious interpretation adopted throughout the revised manuscript.

Abstract:

please revise the opening sentence, which appears to be incomplete in its current form.

Introduction - has been substantially improved. The rationale for selecting strain GAU-93 and the knowledge gap are now more clearly presented.

Materials and Methods this section has been substantially improved. The methodological description is now sufficiently detailed, and the statistical analysis is appropriate for the factorial experimental design

Results

Please carefully revise the formatting of the figures and axis labels. In several graphs, units are not formatted according to standard scientific notation (e.g., g⁻¹, mg g⁻¹, μmol g⁻¹ FW, etc.): exponent should be displayed as a superscript.
Lines 309-311 - "Overall, these results indicate...") : rephrased to remain purely descriptive, leaving interpretation for the Discussion.

Discussion: well aligned with the Results; adopts a much more balanced interpretation of the findings.

minor suggestion: statements referring to improved drought tolerance through flavonoid metabolism could be further moderated to emphasize that the proposed mechanisms remain hypothetical and require further experimental validation.

Conclusions: consistent with the presented data.

minor refinement-- the final statement linking the observed physiological responses to antioxidant and secondary metabolic pathways could be phrased more cautiously - emphasize that these mechanisms remain inferential rather than directly ddemonstrated

Author Response

Comments 1:

Specific comments:

Title: could be slightly moderated to better reflect the associative nature of the evidence presented. The expression "by promoting" implies a causal mechanism that was not directly demonstrated in the present study. A title based on "is associated with" or equivalent wording would be more consistent with the cautious interpretation adopted throughout the revised manuscript.

Response: Thank you for this important suggestion. We agree that the phrase “by promoting” may imply a causal mechanism that was not directly established in the present study. Therefore, we have moderated the title to reflect the associative nature of our findings. The revised title is:

Line 1-4: “Sinorhizobium meliloti GAU-93 Inoculation Is Associated with Reduced Drought-Induced Oxidative Damage and Enhanced Flavonoid Accumulation and Antioxidant Capacity in Alfalfa” was modified.

Comments 2:

Abstract:

please revise the opening sentence, which appears to be incomplete in its current form.

Response: Thank you for pointing this out. We have revised the incomplete opening sentence in the Abstract to improve its grammatical completeness and clarity. The revised sentence reads:

Line 14-15: “This study investigated the effects of Sinorhizobium meliloti GAU-93 inoculation on the growth and drought responses of alfalfa (Medicago sativa L.)” was modified.

Comments 3:

Introduction - has been substantially improved. The rationale for selecting strain GAU-93 and the knowledge gap are now more clearly presented.

Response: We sincerely thank the reviewer for this positive assessment. The reviewer’s previous comments were very helpful in improving the focus, logic, and scientific context of this section.

Comments 4:

Materials and Methods this section has been substantially improved. The methodological description is now sufficiently detailed, and the statistical analysis is appropriate for the factorial experimental design

Response: We sincerely thank the reviewer for recognizing the improvements made to the Materials and Methods section and the appropriateness of the revised statistical analysis for the factorial experimental design.

Comments 5:

Results

Please carefully revise the formatting of the figures and axis labels. In several graphs, units are not formatted according to standard scientific notation (e.g., g⁻¹, mg g⁻¹, μmol g⁻¹ FW, etc.): exponent should be displayed as a superscript.

Response: Thank you for this careful observation; we have thoroughly revised all figures and axis labels to ensure that units follow standard scientific notation, with negative exponents correctly formatted as superscripts (e.g., g⁻¹, mg g⁻¹, and μmol g⁻¹ FW).

Lines 309-311 - "Overall, these results indicate...") : rephrased to remain purely descriptive, leaving interpretation for the Discussion.

Discussion: well aligned with the Results; adopts a much more balanced interpretation of the findings.

Response: We sincerely thank the reviewer for this positive assessment; the statement in Lines 309–311 has been revised to remain purely descriptive.

Line 312-314: “GAU-93-inoculated plants had significantly higher nodule numbers than uninoculated plants at all SWC levels, whereas a significant increase in nodule weight was observed only at 15% SWC” was modified.

minor suggestion: statements referring to improved drought tolerance through flavonoid metabolism could be further moderated to emphasize that the proposed mechanisms remain hypothetical and require further experimental validation.

Response: We thank the reviewer for this valuable suggestion and have further moderated the relevant statements to clarify that the involvement of flavonoid metabolism in GAU-93-associated drought tolerance represents a proposed mechanism that requires further experimental validation.

Line 413-415: “GAU-93 inoculation was associated with changes in selected flavonoid-related metabolites and enzyme activities, although these responses varied among indicators and SWC levels; thus, the potential contribution of flavonoid metabolism to drought tolerance remains hypothetical and requires further experimental validation” was modified.

Conclusions: consistent with the presented data.

minor refinement-- the final statement linking the observed physiological responses to antioxidant and secondary metabolic pathways could be phrased more cautiously - emphasize that these mechanisms remain inferential rather than directly ddemonstrated

Response: Thank you for this helpful suggestion. We have revised the final statement in the Conclusions to clarify that the proposed involvement of antioxidant and secondary metabolic pathways remains inferential and requires further experimental validation.

Line 526-530: “These findings suggest a possible association between GAU-93 inoculation and changes in antioxidant and secondary metabolic pathways; however, the involvement of these pathways in improving drought responses remains inferential and requires further experimental validation” was modified.

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Although limited in its scope, the work is correctly designed, performed and described. The results have an interest from both an eco/physiological and an applicative point of view.

The study has a particular merit in exploring the regulation of phenol metabolism and nodulation under stress. Regrettably, this part could have been stressed further (how were the metabolites and genes selected? what is their biological role?), and discussed in more detail.

The following edits are suggested for a more correct interpretation of data:

Data are presented on fresh weight basis in some figures (3, 4), dry weight in others (5). It would be appropriate to uniform the data, most notably because water content, being directly affected by drought stress, may be a confounding factor.
Paragraph 3.7 and figure 7 do not seem to provide any further insight. Correlation between the tested indexes only demonstrates they have a common driver (i.e. drought, which is obvious), but does not explain the underlying causal links. Plus, it is not relevant to symbiont inoculation. Suggest removing.

It may be worth noting (and discussing) that S. meliloti inoculation does not seem to induce a specific set of plant stress defenses, as shown by the general absence of I x SWC effects. Rather, it contributes to plant stress tolerance by maintaining the physiological function under stress.

Author Response

Comments 1:

The following edits are suggested for a more correct interpretation of data:

Data are presented on fresh weight basis in some figures (3, 4), dry weight in others (5). It would be appropriate to uniform the data, most notably because water content, being directly affected by drought stress, may be a confounding factor.

Response: Thank you for this important and constructive comment. We agree that the use of different biomass bases among figures may complicate direct comparisons and that drought-induced changes in tissue water content may influence measurements expressed on a fresh-weight basis. Ideally, all mass-based measurements would be expressed using a consistent biomass basis. However, paired fresh- and dry-weight measurements were not recorded for each biological replicate at the time of sampling. In addition, the moisture content and dry-matter proportion of the corresponding samples were not determined. Therefore, neither the fresh-weight-based data could be accurately converted to a dry-weight basis nor the dry-weight-based data converted to a fresh-weight basis. The use of an estimated or average conversion factor would be inappropriate because tissue water content varied substantially among the different soil water content treatments and could introduce additional uncertainty or bias into the results.

Accordingly, we retained the original biomass basis used for each measurement rather than performing an unreliable retrospective conversion. The indices presented in Figures 3 and 4 remain expressed on a fresh-weight basis, whereas those in Figure 5 remain expressed on a dry-weight basis, consistent with the original sample preparation and analytical procedures. We have clarified the normalization basis in the Methods, figure axes, and figure legends and have avoided direct quantitative comparisons between variables expressed on different biomass bases.

We also acknowledge that fresh-weight normalization cannot completely exclude the potential influence of drought-induced variation in tissue water content. Therefore, the corresponding results have been interpreted more cautiously, and this limitation has been added to the revised Discussion. Relative water content is presented separately to characterize plant water status under the different soil water content treatments.

Line 484-487: “Because the biochemical measurements were normalized to fresh weight, differences in tissue water content among the SWC treatments may have partially influenced the calculated concentrations. Therefore, these results should primarily be interpreted as relative changes among treatments rather than absolute changes in metabolite accumulation” was added.

Comments 2:

Paragraph 3.7 and figure 7 do not seem to provide any further insight. Correlation between the tested indexes only demonstrates they have a common driver (i.e. drought, which is obvious), but does not explain the underlying causal links. Plus, it is not relevant to symbiont inoculation. Suggest removing.

Response: Thank you for this important comment. We agree that the correlations among the measured indices may largely reflect their shared responses to soil water availability and do not establish causal relationships or specifically explain the effects of GAU-93 inoculation. Therefore, Paragraph 3.7 and Figure 7, together with the corresponding interpretations in the Discussion, have been removed from the revised manuscript. The subsequent figure and all related cross-references have been renumbered accordingly.

Comments 3:

Response: We thank the reviewer for this insightful comment. We agree that the generally non-significant inoculation × SWC interactions do not support the induction of a distinct drought-specific defense program by S. meliloti GAU-93. We have therefore revised the Discussion to emphasize that the effects of inoculation were largely additive across water regimes and were more consistent with the maintenance of plant water status, growth, antioxidant capacity, and secondary metabolic function under stress, rather than with the activation of a specific set of drought defenses. The details are stated as follows:

Line 455-457: “Taken together, these results suggest that GAU-93 may contribute to improved drought responses in alfalfa, although the magnitude of the response varied across traits and SWC levels” was changed to “Notably, the inoculation × SWC interaction was not significant for most measured traits, indicating that GAU-93 did not induce a distinct drought-specific defense response. Instead, its effects were generally additive across water regimes, suggesting that inoculation may support stress tolerance primarily by maintaining plant physiological and metabolic functions under water-limited conditions”.

Line 467-469:“These patterns suggest that rhizobial inoculation may contribute to improved plant water status and growth under the tested conditions” was changed to “These patterns are more consistent with a general maintenance of plant water status and growth across the tested water regimes than with the activation of a specific drought-induced response”.

Line 476-479:“These results suggest that GAU-93 may contribute to redox regulation under water deficit, although the response varied across traits and SWC levels” was changed to “Because significant inoculation × SWC interactions were generally absent, the observed differences in antioxidant traits should not be interpreted as evidence that GAU-93 induced a drought-specific antioxidant defense program. Rather, GAU-93 may help maintain antioxidant capacity and limit oxidative deterioration across different water regimes”.

Line 495-499:“These results suggest that GAU-93 may influence flavonoid-related metabolism under water deficit, but the response remained gene- and treatment-dependent” was changed to “Similarly, the changes in flavonoid-related metabolites, enzyme activities, and gene expression were not associated with a consistent inoculation × SWC interaction, suggesting that GAU-93 may help maintain secondary metabolic activity rather than induce a distinct drought-specific flavonoid response”.

Author Response File: Author Response.docx

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

Round 1

Reviewer 1 Report

This manuscript provides a comprehensive study of the effects of Sinorhizobium meliloti GAU-93 on drought tolerance in alfalfa. The investigation combines several assays, including histochemical, physiological, biochemical, and molecular measurements.

It contains valuable data and addresses a significant topic. However, some issues need to be clarified before publication.

Please find my detailed comments in the attached file.

Comments for author File: Comments.pdf

Reviewer 2 Report

General comments: The manuscript covers an important topic and combine several physiological, biochemical, and molecular approaches to study drought tolerance in alfalfa. However, there are several issues that need to be addressed to improve the clarity and robustness of the study.

since the study evaluates plant responses under different soil water content levels, the use of fresh weight as a growth-related parameter may introduce bias due to variation in tissue water content across treatments. Dry weight would provide a more reliable estimate of true biomass accumulation under drought conditions, and its use should be considered or, at minimum, justified by the authors.
experimental design is not consistently and clearly described: definition of soil water content (SWC) levels and the interpretation of these levels as drought treatments. The rationale for considering 60% SWC as part of a drought gradient is not sufficiently justified, and the definition of the control condition varies across sections of the manuscript. The manuscript inconsistently treats the 60% soil moisture treatment as part of a drought gradient, despite being previously defined as a waterlogged condition. This inconsistency directly affects the interpretation of all physiological and growth-related results.
I have important concerns regarding statistical analysis and data presentation. The study appears to involve a factorial design (inoculation × SWC), yet only one-way ANOVA is reported, and the use of Duncan’s multiple range test is not justified. Graphical representations - violin/boxplot-style figures with n = 4 - raise concerns about appropriateness and interpretability given the limited sample size and unclear representation of variability.
inconsistencies between figures, results text, and discussion, particularly in relation to oxidative stress markers (ROS, H₂O₂, O₂•⁻, and MDA). In several cases, the direction of the reported changes is not clearly aligned between the data presentation and the biological interpretation: weakens the conclusions regarding the effect of GAU-93 on oxidative stress mitigation.
methodological descriptions are frequently incomplete or insufficiently detailed (e.g., inoculum preparation, enzyme assays, HPLC conditions, RNA reverse transcription, and histochemical staining procedures), which limits reproducibility
several sections of the Results include interpretative or mechanistic statements that would be more appropriate for the Discussion, leading to overinterpretation of the data.
Figure legends require substantial improvement, as they currently do not provide sufficient detail to allow independent interpretation of the figures, particularly regarding statistical analysis, data representation, and treatment definitions.
figure labeling and axis descriptions require systematic revision to improve clarity and consistency across the manuscript. Y-axis labels should always include units of measurement, and x-axis labels should be standardized to “Soil Water Content (SWC, %)” rather than generic “treatments (%)

Specific comments:

Section 1 - introduction

generally well written; could be more concise in the description of drought responses, particularly regarding ROS production and antioxidant systems, which are reiterated across multiple sentences
describing flavonoid–antioxidant interactions is somewhat strongly mechanistic for an Introduction and could be slightly toned down to better distinguish established knowledge from hypotheses relevant to the present study
knowledge gap is clearly stated; however, the link between flavonoid metabolism and rhizobial drought tolerance could be more explicitly framed as a hypothesis rather than an implied mechanism

Section 2 Materials and Methods

This section raised several questions that should be addressed:

Line 130 - The text refers to 'strains' (in plural), whereas only strain GAU-93 appears to have been used
Linha 140 - the experimental design appears to be factorial: inoculated vs. non-inoculated plants under four soil water content (SWC) levels (15%, 30%, 45%, and 60%).
1. How were these SWC values determined? Were they calculated based on field capacity? The authors should describe the method used to establish the different SWC levels.
2. it is unclear why 60% SWC was considered a waterlogging treatment. SWC alone does not necessarily indicate waterlogged conditions. Were any measurements of soil redox potential (Eh), oxygen availability, or other indicators of waterlogging performed?
Linha 142-143 – “inoculated plants were supplied with 2 mL of GAU-93 suspension applied to the root zone”
1. How was successful inoculation verified?
2. information regarding inoculum preparation and concentration should be provided.
Line 147: “Four biological replicates were collected per treatment”
1. What was considered the experimental unit? Was each replicate a pot (4 pots per treatment)? If so, were the 3 plants within each pot pooled for analysis? Please clarify, as otherwise there is a risk of pseudoreplication.
L148-164: What specific structural parameters were quantified from SEM observations?
L170-172 – How was TW obtained? At what temperature? for how long were samples incubated?
L179: “POD and APX activities were assessed using guaiacol colorimetry”
1. Why was the same approach used for both POD and APX activity determination?
2. APX activity is commonly determined through ascorbate oxidation monitored at 290 nm. Please justify the methodology adopted instead the use of the specific kit to APX
L183-188 – please provide the extraction volume used for soluble sugar determination (“Fresh samples (0.2 g) were homogenized with….. distilled water”. How was the quantification performed?
Linha 189-193: How was the quantification performed?
L195-199 – Regarding the DAB staining procedure
1. Authors should cite the method used.
2. “Alfalfa leaves were immersed in glass containers containing a solution of diamino benzidine (DAB) (0.1 g of DAB dissolved in 100 mL of sterile distilled water” – Was the pH of the DAB solution adjusted?
3. …..containers were then incubated at room temperature for 24 h – Were the samples incubated in the dark during the 24 h staining period?
4. followed by decolorization of the leaves in 80% (v/v) ethanol for 2 h. – At what temperature was this step performed?
5. Was DAB staining assessed qualitatively, or was image analysis used for quantification?
L201-209 – “The samples (1 g) were weighed (which had been powdered and passed through 1 mm sieve)
1. Was this fresh or dry material? If dry material was used, please describe how it was obtained.
2. HPLC conditions should be described in sufficient detail: detector, column, mobile phase, isocratic or gradient elution, flow rate, column temperature, and quantification procedure.
L211-221 – CHI and CHS activity determinations should be described in more detail.
1. Which method was used?
2. Please provide the units of enzyme activity and explain how enzyme activities were calculated. Were standard curves used?
L228-229 – Description of cDNA synthesis
1. lacks essential methodological details and contains inappropriate terminology (“RNA was inverted”).
2. Sentence “The above extracted RNA was inverted with the following system, the reaction system was as follows: 37℃ 15 min, 85℃ 5 sec, 4℃.” is very difficult to understand. Please rewrite and clarify that RNA was reverse-transcribed into cDNA and provide complete information regarding the reverse transcription system, including enzyme/kit used
L240-247 - The statistical analysis section requires clarification
1. The study appears to follow a factorial design with 2 factors (GAU-93 inoculation and soil water content), yet only one-way ANOVA is reported. Authors should explain how the analyses were performed and consider whether a two-way ANOVA would be more appropriate to evaluate both main effects and their interaction.
2. the definition of the 4 replicates used in the study should be clearly stated, and information regarding verification of ANOVA assumptions would improve the reproducibility of the analysis.

Section 3 – Results

Section 3.1 –

The interpretation of the SEM observations would benefit from more cautious wording. While the images support differences in nodule morphology and internal structural organization among treatments, it is not entirely clear whether the observed structures can be confidently identified as rhizobia cells at the magnifications presented. Please clarify the basis for this interpretation or consider revising the text accordingly.
SEM observations (Fig 1A) are shown only for GAU-93-inoculated plants. How do authors distinguish the effects of bacterial inoculation from those of soil moisture level?
The statistical analysis and lettering system used in Figure 1 requires clarification.
1. The experiment follows a factorial design (GAU-93 inoculation × soil water content), yet the Methods section reports the use of one-way ANOVA. authors should justify this approach and clarify whether a two-way ANOVA was considered to evaluate the main effects and their interaction.
2. Given the relatively small sample size (n = 4), the authors may also consider justify the choice of Duncan's multiple range test, which is generally less conservative than other multiple comparison procedures.
3. The graphical representation in panels B and C appears to resemble a violin/bean plot despite the very small sample size (n = 4). Please clarify which statistic is represented by the central horizontal line and whether the displayed values correspond to means, as stated in the Materials and Methods section.
4. According to the legend, uppercase letters indicate differences "between treatments", but it is not specified whether these comparisons were performed among all eight treatment combinations, or separately within inoculated and non-inoculated plants.
5. Fig 1 - lettering system is difficult to read, please use bigger font. In panel C – authors should confirm the letter Cb and Bb in the control data for SWC 30 and 45%, respectively.
Figure 1A is not explicitly referenced in the Results section and should be cited directly when presenting SEM observations.
the term "ultrastructural observation" may not be appropriate, as the images primarily show nodule morphology and surface features rather than cellular or subcellular ultrastructure.
the percentage increases reported in the text for nodule number and nodule weight do not appear consistent with the values shown in Figure 1
The term "weight" is insufficiently defined throughout the manuscript and figure legends. Authors should clearly indicate whether the reported values correspond to fresh weight or dry weight and provide the corresponding measurement protocol in the M&M section. This distinction is particularly important in a study focused on water availability, as fresh weight may largely reflect tissue water content rather than actual biomass accumulation.

Section 3.2 –

description of drought severity requires clarification. According to the experimental design, plants were maintained at 45% SWC prior to stress imposition. Therefore, it is unclear why 60% soil moisture is discussed as part of a progressive drought treatment. Since 60% represents a higher water availability than the pre-stress condition, it may not constitute a drought treatment. Please clearly define the control condition and the rationale for considering 60%, 45%, 30%, and 15% soil moisture as a drought gradient.
Several statements in Section 3.2 go beyond a description of the results. This section should describe the observed statistical outcomes without inferring physiological mechanisms.: expressions such as “indicating that GAU-93 promotes water retention”, “thereby improving drought resistance”, and “suggesting that the inoculant enhances stem elongation” should be treated more cautiously and moved to the Discussion section. results section should primarily focus on reporting the observed differences among treatments.
L271-273 – “Fresh weight increased consistently in inoculated plants across all drought intensities, with significant improvements observed particularly under moderate and severe stress (15%, 30%, and 45%; Fig. 2B):
1. description of Figure 2B is not fully consistent with the statistical annotations shown in the figure. asterisks indicate significant differences between inoculated and non-inoculated plants at all moisture levels, including 60%.
2. It’s crucial to clearly define the control condition – in this sentence 45% is considered moderate drought while in the abstract is “well-watered control”
273-275: :” Dry weight exhibited a similar pattern and showed the largest enhancement at 60%, indicating that GAU-93 promotes water retention, thereby improving alfalfa's drought resistance (Fig. 2B-C).”
1. A similar pattern??? Please revise this interpretation since dry weight differed significantly between inoculated and non-inoculated plants only at 60% SWC.
2. 275-278:” The statement that the height advantage was "especially pronounced" under severe drought (15% SWC) does not appear to be directly supported by Figure 2D. The magnitude of the difference between inoculated and non-inoculated plants seems comparable across all SWC, and no statistical comparison of the inoculation effect among moisture treatments is presented. The authors should either provide supporting statistical evidence or moderate this statement.
L278-282 and Fig. 2E
1. RWC is defined in the Methods as a % yet the y-axis appears to be presented on a 0–1 scale rather than 0–100%. Please clarify whether the values represent fractions or % to ensure consistency between the Methods, figure axis and units reported.
2. Given n = 4, the apparent overlap among individual observations in some treatments, and the use of boxplot-style graphics despite the Methods stating that data are presented as means, the authors should clarify exactly what is represented in the figure (median, quartiles, individual observations, mean ± error, etc.) and provide sufficient information to support the reported statistical differences.

section 3.3

this study combines histochemical staining (NBT and DAB) with biochemical quantification to assess ROS accumulation. NBT and DAB staining provide useful in situ visualization of O₂•⁻ and H₂O₂ distribution in leaves, while biochemical assays allow quantitative measurement of ROS, lipid peroxidation, and key metabolites.
Linhas 294-309:
1. “…analysis revealed that under drought treatment, the accumulation of O₂•⁻ in leaves of alfalfa plants inoculated with GAU-93 was significantly higher than that in the control at all treatments groups (Figure 3A-D)”; The description of these results is not entirely clear and requires revision, as the interpretation seems to contradict the trends shown in the data and in the discussion.
2. “…the accumulation of H₂O₂ in leaves of alfalfa plants inoculated 301 with GAU-93 was significantly higher than control in the 30%+GAU-93, 45%+GAU-93 and 302 60%+GAU-93 treatments (Figure 3B-C).”; The description of these results is not entirely clear and requires revision, as the interpretation seems to contradict the trends shown in the data and in the discussion

section 3.4

“Both GAU-93 and control showed significant increases in CAT, APX, SOD and POD activities as drought stress increased“ - The interpretation of the SWC gradient is unclear, as the directionality of the physiological responses suggests that higher SWC corresponds to less stress conditions and the enzyme activities showed enhanced value from 15% to 60% SWC.

section 3.5

L335 – “Meanwhile, 15%+GAU-93, 45%+GAU-93 and 60%+GAU-93 treatment showed higher CHS and CHI activities than 15%+control, 45%+control and 60%+control treatment (Fig. 5C-D)”. – I cannot see differences between GAU-93 and Control for SWC 15% in CHI activity!
“These results suggest that GAU-93 treatment could increase endogenous daidzein and 340 genistein level and related enzyme activity, thereby enhancing alfalfa's drought tolerance.” – Move to the discussion section

section 3.6

The current description overstates the transcriptional response by implying uniform upregulation of all genes, despite clear evidence of gene- and treatment-specific variability. This lack of precision may lead to an overestimation of the extent of GAU-93-induced molecular regulation

Section 3.7

Ggiven the strong influence of tissue water content on fresh weight under drought stress, the use of FW in correlation and Mantel analyses may lead to influenced or inflated associations between biomass and physiological traits. Re-analysis using dry weight would likely provide a more stable and biologically meaningful representation of trait relationships.

Sections 4 and 5

the inconsistencies and overinterpretation identified in the Results section also compromise several conclusions presented in the Discussion, which should be carefully revised to ensure that all mechanistic claims are directly supported by the corrected data presentation and statistical interpretation.