Phenolic Composition and Antioxidant Capacity of Pistachio Seed Coats at Different Tree Ages Under Saline Irrigation Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, authors analyzed phenolic components and antioxidant capacity in Pistachio seed coats from different ages pistachio trees planted in 2002, 2009, and 2011 that were irrigated with high-salinity water containing traces of boron (B) and selenium (Se). The results indicated that tree age significantly influenced total antioxidant capacity and phenolic content, and HPLC identified one phenolic acid (gallic acid) and nine flavonoids, with Catechin, procyanidin B1, and eriodyctiol showing the highest abundant in the oldest trees. This study comprehensively evaluated polyphenolic composition and antioxidant capacity in pistachio seed coats across different ages under saline irrigation conditions. These findings contribute to advancing scientific research in this field, however, there are many issues that should be addressed to facilitate reading and make the information useful:

1. The study was conducted in open fields and may be subject to extreme weather (such as high temperatures, heavy rains) or pest interference.At the same time, the experiment did not explicitly exclude interfering factors such as the initial physical and chemical properties of soil and the initial growth state of trees (such as the difference in the initial seedling condition of trees planted in 2002/2009/2011). This "single variable attribution" directly attributes the "positive correlation between tree age and phenolic content" to the "dominant role of tree age", without excluding the "interaction effect of tree age and soil nutrient accumulation", and may ignore the complex field interaction. Based on the key findings of this study, it is suggested that the authors revise the title to "Phenolic Composition and Antioxidant Capacity of Pistachio Seed Coats at Different Tree Ages Under Saline Irrigation Conditions".
2. The author is requested to supplement the research progress on the salt tolerance mechanism of pistachios in the introduction, especially the role of boron and selenium minerals in the accumulation of endogenous substances in pistachios and their relationship with stress resistance.
3. In the method, the author only pointed out the conductivity of saline and mineral boron and selenium, and did not explain the other ion salts in saline.The author was asked to introduce the salt composition of irrigation saline in the method.
4. Please provide the basis for the high-low salt setting. Based on the test results, the authors are requested to explain why there was no significant difference in total antioxidant capacity and total phenolic content between high-salt and low-salt irrigation treatments. Additionally, the authors are requested to supplement the antioxidant capacity and total phenolic content of pistachios under normal growth conditions in the no-salt treatment.
5. The study investigates the effects of tree age and salt stress on polyphenol accumulation in fruit peels, with the authors attributing phenolic accumulation to tree age. However, the authors only measured data from three tree age groups. It is suggested that the authors supplemented the findings with additional data, such as corresponding measurements from 2023 and 2024.
6. It is suggested that the authors placedFigure S1 in the main text.

Author Response

Answer to Reviewers

We would like to thank all reviewers for taking the time to read and evaluate our manuscript and for making concrete and insightful comments that helped to improve the quality of our manuscript. The revised manuscript has a Track-Changes option, so that you can see exactly what was changed.  The pdf document is without the Track-Changes option to allow for more unobstructed reading. We use the line numbering and pages of the PDF to describe the location of  concrete changes listed below.

Overview on most substantial changes based on all four reviews

 

In revising the manuscript, we made several changes in response to the reviewers' comments. The most important scientific update concerns the statistical analysis. The original version used one-factor Kruskal–Wallis tests, which evaluated irrigation treatment independently of planting year. We replaced this with a two-factor aligned rank transform ANOVA (with comparison to a parametric two-way ANOVA), which more appropriately reflects the factorial structure of the experiment. Under this analysis, irrigation treatment effects could be evaluated while accounting for the influence of planting year. As a result, the salinity responsiveness of quercetin, isoquercetin, and also gallic acid became more apparent; gallic acid had been borderline under the one-factor approach, and the updated analysis resolved its smaller treatment effect more clearly.

We also reorganized the manuscript by separating the combined “Results and Discussion” section into two distinct sections, which required adjusting the order and flow of several passages. The Introduction now includes additional background on ion exclusion, compartmentalization, osmotic adjustment, and the potential roles of boron and selenium in pistachio responses to saline irrigation. The Discussion was expanded with paragraphs addressing the roles of quercetin, isoquercetin, and gallic acid, as well as a new subsection outlining study limitations and directions for future work. Finally, we clarified the study’s contribution in the Introduction and Conclusions and updated several references, including those concerning the Folin–Ciocalteu assay and boron/selenium-mediated stress responses.

 

Reviewer 1:

 

Comment 1: The study was conducted in open fields and may be subject to extreme weather (such as high temperatures, heavy rains) or pest interference. At the same time, the experiment did not explicitly exclude interfering factors such as the initial physical and chemical properties of soil and the initial growth state of trees (such as the difference in the initial seedling condition of trees planted in 2002/2009/2011). This "single variable attribution" directly attributes the "positive correlation between tree age and phenolic content" to the "dominant role of tree age", without excluding the "interaction effect of tree age and soil nutrient accumulation", and may ignore the complex field interaction. Based on the key findings of this study, it is suggested that the authors revise the title to "Phenolic Composition and Antioxidant Capacity of Pistachio Seed Coats at Different Tree Ages Under Saline Irrigation Conditions".

Response 1: We thank the reviewer for this suggestion. We have revised the title exactly as recommended:

New Title
“Phenolic Composition and Antioxidant Capacity of Pistachio Seed Coats at Different Tree Ages Under Saline Irrigation Conditions.”

To address the reviewer’s concern about uncontrolled environmental factors, we added the following text to the Discussion (Line 583 p. 16, Section 4.5 Study Limitations and Perspectives):

“Field-based sampling is also influenced by environmental variability, including seasonal weather conditions, pest pressure, and soil heterogeneity, which may contribute to sample variability.”

Comment 2: The author is requested to supplement the research progress on the salt tolerance mechanism of pistachios in the introduction, especially the role of boron and selenium minerals in the accumulation of endogenous substances in pistachios and their relationship with stress resistance.

Response 2: We expanded the Introduction by adding two full paragraphs describing pistachio salinity tolerance mechanisms and the roles of B and Se.
The following text appears in the revised manuscript (p. 2, Introduction, paragraphs beginning line 64 and line 78):

“Pistachio trees rely on several coordinated physiological mechanisms to maintain productivity under saline irrigation. Key among these are xylem ion exclusion and ion retrieval, which restrict the movement of sodium and chloride into photosynthetic tissues and favor their storage in roots and woody stems [6,12]. Both controlled root-stock studies and field observations show that pistachio can retain substantial amounts of Na⁺ and Cl⁻ in roots and basal stems while transporting comparatively smaller quantities to leaves, and that more tolerant rootstocks such as Pistacia atlantica maintain more favorable K⁺/Na⁺ and Ca²⁺/Na⁺ ratios in foliage under saline irrigation [5,6,12]. Pistachio also employs intracellular compartmentalization and osmotic adjustment, including increases in proline and soluble sugars under salt stress, which support water balance and limit oxidative injury [7,9]. These ion-management and metabolic responses, together with the buffering capacity of a large perennial woody biomass, help explain why pistachio generally tolerates higher salinity than many other nut crops, even though the exact efficiency of these mechanisms varies among root-stocks.

Micronutrients that commonly accompany salinity in drainage waters in the westside of California’s SJV, particularly B and Se, can further influence these responses. Moderate B levels support membrane stability, cell-wall function, and anti-oxidant activity [17], and B amendments have been shown to enhance glycine betaine accumulation, and reduce salt-induced leaf damage in pistachio seedlings [11]. Although Se has not been tested directly in pistachio, work in soybean and other crops shows that low concentrations of Se or Se+B can strengthen antioxidant defenses, im-prove leaf hydration, and reduce oxidative damage during salt exposure [18,19]. At non-toxic concentrations, both B and Se can promote the accumulation of endogenous protective metabolites, including proline, glycine betaine, and soluble sugars, and may therefore contribute positively to the overall resilience of pistachio orchards irrigated with moderately saline drainage waters.”

 

Comment 3: In the method, the author only pointed out the conductivity of saline and mineral boron and selenium, and did not explain the other ion salts in saline. The author was asked to introduce the salt composition of irrigation saline in the method.

Response 3: We added the composition of both irrigation waters.
The added text appears in the Field Design subsection (p. 4, starting line 159):

“The two natural water sources in the westside of California’s SJV differed markedly in ionic composition. The canal water contained 25–66 mg Cl L⁻¹, 31–72 mg Na L⁻¹, 11–41 mg Ca L⁻¹, 5–18 mg Mg L⁻¹, and 9–55 mg S L⁻¹ (pH 7.5–8.0), while the drainage water contained 575–950 mg Cl L⁻¹, 356–670 mg Na L⁻¹, 218–370 mg Ca L⁻¹, 56–112 mg Mg L⁻¹, and 270–545 mg S L⁻¹ (pH 7.8–8.4). The high-saline water also contained higher concentrations of B (4–9 mg B L⁻¹) and Se (75–150 µg Se L⁻¹), whereas the low-saline water contained 0.5–1.0 mg B L⁻¹ and 5–10 µg Se L⁻¹. These differences reflect the elevated ionic strength and salt load typical of reclaimed irrigation waters in the SJV.”

Comment 4: Please provide the basis for the high-low salt setting. Based on the test results, the authors are requested to explain why there was no significant difference in total antioxidant capacity and total phenolic content between high-salt and low-salt irrigation treatments. Additionally, the authors are requested to supplement the antioxidant capacity and total phenolic content of pistachios under normal growth conditions in the no-salt treatment.

Response 4: We added several clarifications in the Introduction and Discussion.

(i) Basis for high vs low salinity setting:

Added to Introduction (p. 3, near line 131):

“The drainage water, typical of the westside of the SJV, contains naturally elevated sodium and chloride concentrations that are approximately tenfold higher than the good-quality canal water available for purchase when supplies permit, and reflects the saline conditions under which drought-susceptible orchards on the westside of the SJV were originally managed.”

(ii) Why no significant changes in antioxidant capacity:

Added text in Discussion (p. 11, near line 382):

“Despite the large ionic contrast between drainage water and canal water, antioxidant capacity remained statistically unchanged across irrigation treatments. This indicates that the seed coat, already known to be a rich source of reducing agents and radical scavengers, maintains a robust and resilient antioxidant reservoir even after prolonged exposure to high salinity.”

Added text in Discussion (p. 16, near line 567)

“Total antioxidant capacity remained unchanged, suggesting that pistachio relies on adjustments in specific compounds rather than expanding the overall antioxidant pool. These patterns align with pistachio’s established strategies for regulating Na⁺ and Cl⁻ through root-level exclusion, controlled xylem loading, and vacuolar sequestration, which together limit ion-induced oxidative pressure.”

(iii) Regarding absence of a no-salt control

 

We appreciate the reviewer’s request to include antioxidant capacity and total phenolic content for pistachios grown under normal, non-saline conditions. Unfortunately, the long-term field experiment did not include a true no-salt control group; the comparison was designed around the two available irrigation sources in commercial practice—saline drainage water and low-salinity canal water. Because the field site is no longer accessible to us, it is not possible to obtain additional no-salinity samples. We also considered drawing on published values; however, reported phenolic and antioxidant levels vary widely across studies due to differences in cultivar, climate, soil chemistry, irrigation, and post-harvest handling, and these conditions are generally not matched to our field system. For these reasons, supplementing the manuscript with no-salinity baseline values from other reports would not allow for a consistent comparison with our field data.

Comment 5: The study investigates the effects of tree age and salt stress on polyphenol accumulation in fruit peels, with the authors attributing phenolic accumulation to tree age. However, the authors only measured data from three tree age groups. It is suggested that the authors supplemented the findings with additional data, such as corresponding measurements from 2023 and 2024.

Response 5: We cannot obtain additional years of samples, and the field site is no longer accessible. So we address this limitation directly in our Discussion section in a new subsection entitled “4.5 Study Limitations and Perspectives”.

Added text in Discussion (p. 16, starting line 578)

“This study focused on a single harvest season and did not include direct measurements of ion accumulation or other physiological indicators of salt stress such as sodium, chloride, or oxidative stress markers. Because only the seed coat was analyzed, potential metabolic adjustments in other tissues—such as leaves, hulls, or roots—remain unresolved. The dataset therefore provides a biochemical snapshot of a long-term field system rather than a temporal profile of salinity adaptation. Field-based sampling is also influenced by environmental variability, including seasonal weather conditions, pest pressure, and soil heterogeneity, which may contribute to sample variability. Multi-year sampling that couples ion analysis with tissue-wide metabolite profiling will be needed to link phenolic composition more directly to physiological and agronomic outcomes.”

Comment 6: It is suggested that the authors placed Figure S1 in the main text.

Response 6: We have moved Figure S1 into the main text as Figure 1.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear editor and authors

The present study is part of a long-term investigation of saline irrigation in mature pistachio orchards of different ages located in the Grassland Basin Water District near Firebaugh, California, USA. The main objective of the present work was to study the effect of two irrigation conditions on the content of antioxidants in the seed coat of pistachio seeds. For the study, trees planted in 2002, 2009, and 2011 that had been irrigated with drainage ("high-salinity") water were used for a three-year experiment, which began in 2018. The assay consisted in two irrigation conditions: irrigation with low-salinity water and continuation of irrigation with drainage water. Three years later, nuts form both treatments were harvested to evaluate how irrigation quality affected the concentration of antioxidants in seed coats (the main site of antioxidants accumulation in pistachio seeds). The main traits evaluated were total antioxidant capacity (by three classical procedures), total phenolics content (by the Folin-Ciocalteu method) and changes in the concentration of specific phenolics (mostly flavonoids), determined by HPLC.

In general terms the manuscript is clear and well written.

My main concern has to do with two key aspects:

- As described and considering the way the results are presented, it is not clear to me how was the statistical analysis performed (basically how were the samples of the different experimental conditions grouped to assess the effects of the two factors being evaluated: year of sowing and irrigation treatment). This is because a nonparametric “one factor” test was used, when a two-factor statistical test would have been more appropriate.

- Aside from the traits above mentioned, it was not assessed whether the irrigation treatments actually affected sodium levels or oxidative stress markers in the seed coat. This would have been important to confirm whether the reported lack of differences between treatments was actually due to the fact that the tissue under study did not experience a saline stress condition, or whether stress tolerance was due to another physiological mechanism that does not involve an increase in the synthesis of antioxidant metabolites (and particularly flavonoids) in the seed coat, as expected.  If such measurements cannot be performed and included, at least, a discussion on these questions has to be incorporated. Finally, the F-C method for the determination of total phenolics should not be considered specific as indicated.

Please see the revised pdf file attached for detailed comments on the text.

Comments for author File: Comments.pdf

Author Response

Answer to Reviewers

 

We would like to thank all reviewers for taking the time to read and evaluate our manuscript and for making concrete and insightful comments that helped to improve the quality of our manuscript. The revised manuscript has a Track-Changes option, so that you can see exactly what was changed.  The pdf document is without the Track-Changes option to allow for more unobstructed reading. We use the line numbering and pages of the PDF to describe the location of concrete changes listed below.

Overview on most substantial changes based on all four reviews

 

In revising the manuscript, we made several changes in response to the reviewers' comments. The most important scientific update concerns the statistical analysis. The original version used one-factor Kruskal–Wallis tests, which evaluated irrigation treatment independently of planting year. We replaced this with a two-factor aligned rank transform ANOVA (with comparison to a parametric two-way ANOVA), which more appropriately reflects the factorial structure of the experiment. Under this analysis, irrigation treatment effects could be evaluated while accounting for the influence of planting year. As a result, the salinity responsiveness of quercetin, isoquercetin, and also gallic acid became more apparent; gallic acid had been borderline under the one-factor approach, and the updated analysis resolved its smaller treatment effect more clearly.

We also reorganized the manuscript by separating the combined “Results and Discussion” section into two distinct sections, which required adjusting the order and flow of several passages. The Introduction now includes additional background on ion exclusion, compartmentalization, osmotic adjustment, and the potential roles of boron and selenium in pistachio responses to saline irrigation. The Discussion was expanded with paragraphs addressing the roles of quercetin, isoquercetin, and gallic acid, as well as a new subsection outlining study limitations and directions for future work. Finally, we clarified the study’s contribution in the Introduction and Conclusions and updated several references, including those concerning the Folin–Ciocalteu assay and boron/selenium-mediated stress responses.

Reviewer 2:

 

My main concern has to do with two key aspects:

Major concern 1: As described and considering the way the results are presented, it is not clear to me how was the statistical analysis performed (basically how were the samples of the different experimental conditions grouped to assess the effects of the two factors being evaluated: year of sowing and irrigation treatment). This is because a nonparametric “one factor” test was used, when a two-factor statistical test would have been more appropriate.

Response to major concern 1: We thank the reviewer for raising this important point.

We replaced the earlier one-factor Kruskal–Wallis approach with a two-factor aligned rank transform ANOVA (ART ANOVA), which evaluates irrigation treatment and planting year simultaneously. This change is now fully reflected in both the main text and the Supplement.

Data Analysis subsection (p. 6, around lines 246):

“We tested for normal distribution (Shapiro-Wilk test) and equal variances (Levene’s test).  Because multiple variables deviated from normality and two showed unequal variances, we analyzed all responses using a two-way aligned rank transform ANOVA (ART ANOVA). Sidak-adjusted pairwise comparisons were used to test differences among factor levels. Parametric two-way ANOVA yielded equivalent conclusions which is presented in the Supplement.”

Correspondingly, the Results section now reports the ART ANOVA outcomes. For example, in Section 3.1 (p. 6, around line 258):

“According to the results of statistical tests from Table S1, no significant differences were observed for seed coat samples from trees with different saline irrigation treatments (all p > 0.025). Only the planting year (e.g., 2002, 2009, 2011) was a significant factor (p < 0.0001). The interaction term between treatment and planting year was not significant (p > 0.13).”

Figures and captions were updated accordingly. We also revised the Supplement to provide a more transparent description of the statistical workflow, including normality and variance tests, the ART ANOVA model, the comparison to parametric ANOVA, and the full Sidak post-hoc results. These additions address the reviewer’s request for clearer documentation of how the two-factor analysis was carried out.

 

Major concern 2: Aside from the traits above mentioned, it was not assessed whether the irrigation treatments actually affected sodium levels or oxidative stress markers in the seed coat. This would have been important to confirm whether the reported lack of differences between treatments was actually due to the fact that the tissue under study did not experience a saline stress condition, or whether stress tolerance was due to another physiological mechanism that does not involve an increase in the synthesis of antioxidant metabolites (and particularly flavonoids) in the seed coat, as expected.  If such measurements cannot be performed and included, at least, a discussion on these questions has to be incorporated.

Response to major concern 2: We agree with the reviewer that sodium concentrations and oxidative stress markers would provide additional physiological context. These measurements were not included in the current study and would require separate extraction protocols and/or analytical techniques from those used here. Because the present work focuses on phenolic composition in the seed coat, we have not pursued these measurements at this time. Instead, we now explicitly acknowledge this point in the Study Limitations section.

Added text in Discussion (p. 16, starting line 578)

“This study focused on a single harvest season and did not include direct measurements of ion accumulation or other physiological indicators of salt stress such as sodium, chloride, or oxidative stress markers. Because only the seed coat was analyzed, potential metabolic adjustments in other tissues—such as leaves, hulls, or roots—remain unresolved.”

Major concern 3: Finally, the F-C method for the determination of total phenolics should not be considered specific as indicated.

Response to major concern 3: We thank the reviewer for pointing this out. We revised the manuscript to clarify that the Folin–Ciocalteu assay is not specific to phenolics.

The following exact text was added to Section 4.1 (p. 11, near line 399):

“It is important to note that the FC method does not exclusively detect phenolic com-pounds, as other reducing substances can also contribute to color development [47,48]. Therefore, the FC values represent the overall reducing capacity of the extracts rather than phenolics alone. Moreover, different phenolic compounds do not respond equally in the FC assay; purified standards produce varying color intensities at identical molar concentrations [48,49].”

We also removed the earlier wording in the Method section and Abstract implying that FC specifically quantifies “total phenolics.”

Reviewer 2 provided more detailed comments directly on the manuscript text (PDF):

Detailed comment 1 – from PDF: Please indicate in Figure S1 how the subplots were located (where is high and where is low saline?)

Response to comment 1: Following Reviewer 1’s parallel suggestion, we moved Figure S1 into the main text (now Figure 1) and clarified the locations of the high- and low-salinity sub-blocks in the new figure caption.

“Figure 1. Photograph showing the orchard with the research field blocks marked in yellow. The southern half of the blocks, closer to the canal, received the low-saline water.”

Detailed comment 2 – from PDF: FC: The F-C method is not specific for total phenolics determination, since different compounds may also react and contribute to color development (e.g. Sánchez-Rangel et al., 2013:  DOI:10.1039/c3ay41125g; Bastola et al, 2017: DOI: 10.4236/ajac.2017.86032). This should be taken into account when discussing the results obtained.

Response to comment 2: Please, see answer to major concern 3. We incorporated the suggested references [47,48].

Detailed comment 3 – from PDF: Please, see comment regarding the statistical analysis. It is not clear to me how the statistical analysis was performed. According to the experimental design, a two-way ANOVA should have been performed. Hence, how were the effects of the two factors tested by the one way Kruskal-Wallis non-parametric test? It is not clear in the graphs I and J which treatments differ significantly, since, as indicated, it appears that, for both compounds, the significant difference occurs between the high and low saline treatments of the 2009 samples...Please check also the other figures

Response to comment 3: Please, see answer to major concern 1. We updated the statistical method (ART ANOVA with Sidak contrasts) and revised the figures accordingly. Details on the statistical analysis for all ten phenolic compounds are available in the revised Supplement (Table S3).

Detailed comment 4 – from PDF: Conclusion: The lack of significant differences between salinity treatments for nearly all the phenolics analized may reflect the existence of other physiological strategies aimed at increasing salt tolerance. This should have been taken into account when discussing the data. In this sense, it would have been important to quantify the amount of (at least) sodium and some indicator of oxidative stress in the samples, in order to determine if the implemented treatments actually induced a relevant physiological change that could have justified, for example, an increase in the antioxidant metabolism (and particularly in C investment for flavonoids synthesis) in the analyzed tissue.

Response to comment 4 We agree and expanded the Discussion to address this point. As mentioned above we acknowledge the lack of sodium data as a limitation of our study. The following text, now in Section 4.4 (p.16 near line 567), addresses alternative salinity-tolerance mechanisms in pistachio:

“Total antioxidant capacity remained unchanged, suggesting that pistachio relies on adjustments in specific compounds rather than expanding the overall antioxidant pool. These patterns align with pistachio’s established strategies for regulating Na⁺ and Cl⁻ through root-level exclusion, controlled xylem loading, and vacuolar sequestration, which together limit ion-induced oxidative pressure. When considered alongside these ion-homeostasis mechanisms, the compound-specific responses observed here offer in-sight into how pistachio maintains seed-coat integrity under saline irrigation.”

Reviewer 3 Report

Comments and Suggestions for Authors

In this study the authors evaluate the total antioxidant capacity of seed coat extracts and quantified specific phenolic compounds to examine how metabolite levels varied with tree age under irrigation with low and high saline water. The results of this research can be important for sustain irrigated agriculture in California and other regions with similar conditions. However, in order to be published I consider that the manuscript needs several improvements. All detailed comments and suggestions can be found bellow:

- I recommend you to present the keywords in alphabetical order.

- Transfer the text from lines 103-109 to M&M section, subsection Field Design. Also, I recommend you to insert the information from lines 118-127 in the Field Design subsection and remove the Pistachios (line 117) as subsection title.

- Transfer the text from lines 127-130 to subsection “Seed coat extract preparation”.

-According to the journal’s requirements (Research Manuscript Sections), you must split the text from section 3 (Results and Discussion) in two separate sections (3. Results; 4. Discussion).

- Reformulate the title of Figure 1, as follows:

Antioxidant capacity assays [ABTS (A), DPPH (B), FRAP (C)] and FC (D) assay

for pistachio seed coat from different age trees under saline treatments.

Each circle on the violin plot represents an individual pistachio seed coat extract (blue = low-saline recovery, orange = continued high-saline irrigation). P-values (according to Bonferroni test) for pairs with significant differences are displayed on the plot.

Each circle on the violin plot represents an individual pistachio seed coat extract (blue = low-saline recovery, orange = continued high-saline irrigation). P-values (according to Bonferroni test) for pairs with significant differences are displayed on the plot.

- Reformulate the title of Table S1, as follows: Statistical tests for antioxidant capacity and FC assays.

- Remove the text „The results from the statistical analysis are summarized in Supplemental Table S1.’ from line 213-214, and reformulate the text from line 214-215 as follows: According with the results of statistical test from Table S1, no significant  differences were observed for seed coat samples from trees with different saline irrigation  treatments.

- Line 231 remove „μmol Trolox equivalents per gram seed coat” and use only μmol Teq/g. Also, remove „μmol gallic acid equivalents (GAeq) per gram seed coat” from lines 240-241 and use only μmol GAeq/g.

- I recommend you to insert Figure S2 (which is representative for its content) in to main text, subsection 3.1.

- Reformulate the text from lines 344-346 (footer of Figure 3), as follows: P-values (according to Bonferroni test) for pairs with significant differences are displayed on the plot.

- Transfer the text from subsection 3.2 and the Figure 2, to Discussion section. In the mentioned (3.2) subsection you can present the results of phenolic compounds from your study.

-Reformulate the title of subsection 3.3, as follows: Effects of planting year and saline irrigation treatment on individual phenolic compounds. or Variation of individual phenolic compounds under the effects of planting year and saline  irrigation treatment.

- The correlations (presented in subsection 3.4) are significant? If so, specify which of them.

- The values of R² from lines 418, 424, represents the coefficient of determination and not the correlation coefficient (which is equal with the square root of these values).

- Considering that PC3 and PC4 express only 21.6% of the phenolic compounds variability, graphs C and D from Figure S4 can be removed.

-The Discussion part (section) can be extended/improved.

- In the Conclusions the perspective part is missing; please describe more accurate perspective statements for directing further studies.

Author Response

Answer to Reviewers

 

We would like to thank all reviewers for taking the time to read and evaluate our manuscript and for making concrete and insightful comments that helped to improve the quality of our manuscript. The revised manuscript has a Track-Changes option, so that you can see exactly what was changed.  The pdf document is without the Track-Changes option to allow for more unobstructed reading. We use the line numbering and pages of the PDF to describe the location of concrete changes listed below.

Overview on most substantial changes based on all four reviews

 

In revising the manuscript, we made several changes in response to the reviewers' comments. The most important scientific update concerns the statistical analysis. The original version used one-factor Kruskal–Wallis tests, which evaluated irrigation treatment independently of planting year. We replaced this with a two-factor aligned rank transform ANOVA (with comparison to a parametric two-way ANOVA), which more appropriately reflects the factorial structure of the experiment. Under this analysis, irrigation treatment effects could be evaluated while accounting for the influence of planting year. As a result, the salinity responsiveness of quercetin, isoquercetin, and also gallic acid became more apparent; gallic acid had been borderline under the one-factor approach, and the updated analysis resolved its smaller treatment effect more clearly.

We also reorganized the manuscript by separating the combined “Results and Discussion” section into two distinct sections, which required adjusting the order and flow of several passages. The Introduction now includes additional background on ion exclusion, compartmentalization, osmotic adjustment, and the potential roles of boron and selenium in pistachio responses to saline irrigation. The Discussion was expanded with paragraphs addressing the roles of quercetin, isoquercetin, and gallic acid, as well as a new subsection outlining study limitations and directions for future work. Finally, we clarified the study’s contribution in the Introduction and Conclusions and updated several references, including those concerning the Folin–Ciocalteu assay and boron/selenium-mediated stress responses.

Reviewer 3:

 

Comment 1: I recommend you to present the keywords in alphabetical order.

Response 1: We re-ordered the keywords as suggested.

Comment 2:  Transfer the text from lines 103-109 to M&M section, subsection Field Design. Also, I recommend you to insert the information from lines 118-127 in the Field Design subsection and remove the Pistachios (line 117) as subsection title.

Response 2: Thank you for these suggestions. We changed the subsection title “Pistachios” into “Field Design” and moved the very technical descriptions from lines 103-109 into the “Field Design” subsection of the M&M section.  Additional changes were made to these manuscript parts since Reviewer 1 had asked to clarify the settings for the low- and high-saline conditions and to provide details on the composition of the irrigation waters.   

Comment 3:  Transfer the text from lines 127-130 to subsection “Seed coat extract preparation”.

Response 3: We transferred the text as requested.

Comment 4: According to the journal’s requirements (Research Manuscript Sections), you must split the text from section 3 (Results and Discussion) in two separate sections (3. Results; 4. Discussion).

Response 4: Thank you for pointing out this important requirement. Our revised manuscript now has two separate section: 3. Results and 4. Discussion

Comment 5: Reformulate the title of Figure 1, as follows:

Antioxidant capacity assays [ABTS (A), DPPH (B), FRAP (C)] and FC (D) assay for pistachio seed coat from different age trees under saline treatments. Each circle on the violin plot represents an individual pistachio seed coat extract (blue = low-saline recovery, orange = continued high-saline irrigation). P-values (according to Bonferroni test) for pairs with significant differences are displayed on the plot.

Response 5: We adopted the reviewer’s revised caption with one modification:
Because the statistical method was changed to ART ANOVA with Sidak, we replaced “Bonferroni” with “Sidak”.

Comment 6:  Reformulate the title of Table S1, as follows: Statistical tests for antioxidant capacity and FC assays.

Response 6: We revised the title of Table S1 as suggested. The new title is more professional.

Comment 7:  Remove the text „The results from the statistical analysis are summarized in Supplemental Table S1.’ from line 213-214, and reformulate the text from line 214-215 as follows: According with the results of statistical test from Table S1, no significant  differences were observed for seed coat samples from trees with different saline irrigation  treatments.

Response 7: We revised the sentence as follows: “According to the results of statistical tests from Table S1, no significant differences were observed for seed coat samples from trees with different saline irrigation treatments (all p > 0.025).”

Comment 8: Line 231 remove „μmol Trolox equivalents per gram seed coat” and use only μmol Teq/g. Also, remove „μmol gallic acid equivalents (GAeq) per gram seed coat” from lines 240-241 and use only μmol GAeq/g.

Response 8: We removed the longer phrases „μmol Trolox equivalents per gram seed coat” and „μmol gallic acid equivalents (GAeq) per gram seed coat” from our manuscript as suggested. We changed all units into μmol Teq/g and μmol GAeq/g (figures and text).

Comment 9:  I recommend you to insert Figure S2 (which is representative for its content) in to main text, subsection 3.1.

Response 9: After evaluation, we did not move Figure S2 into the main text, because doing so would have duplicated information from Figure 1. We already transferred another Figure (Figure S1) into the main text.  So, instead, we added a new sentence clarifying the correlation results in the text (starting p. 6, around line 269):

“All three antioxidant capacity assays (ABTS, DPPH, FRAP) and the FC assay showed very strong and highly significant pairwise correlations (p < 0.001). Pearson coefficients were 0.92 for DPPH and ABTS, 0.92 for DPPH and FC, 0.90 for DPPH and FRAP, 0.88 for ABTS and FRAP, 0.88 for ABTS and FC, and 0.78 for FRAP and FC. An analysis of the correlation coefficients separated by planting year can be found in Supplementary Figure S1.”

Comment 10: Reformulate the text from lines 344-346 (footer of Figure 3), as follows: P-values (according to Bonferroni test) for pairs with significant differences are displayed on the plot.

Response 10: We adopted the reviewer’s wording but replaced “Bonferroni” with “Sidak” to reflect the updated statistical analysis.

Comment 11: Transfer the text from subsection 3.2 and the Figure 2, to Discussion section. In the mentioned (3.2) subsection you can present the results of phenolic compounds from your study.

Response 11: The comparative literature analysis and former Figure 2 are now part of Section “4.2 Phenolic Composition in the Context of Previous Studies”. Figure 2 is now labeled Figure 5 in the revised manuscript.

Comment 12: Reformulate the title of subsection 3.3, as follows: Effects of planting year and saline irrigation treatment on individual phenolic compounds. or Variation of individual phenolic compounds under the effects of planting year and saline  irrigation treatment.

Response 12: We selected the reviewer’s first suggested title.

“3.2 Effects of Planting Year and Saline Irrigation Treatment on Individual Phenolic Compounds”

Comment 13: The correlations (presented in subsection 3.4) are significant? If so, specify which of them.

Response 13: We added p-values after applying the R function cor.test() to show the significance level of the correlations. Below is some sample text from the newly labeled subsection 3.3 Correlation and Cluster Analyses:

“Pearson correlation analysis (Figure 4A) revealed several strong and highly significant relationships among the ten detected phenolic compounds. The three most abundant flavonoids (catechin, procyanidin B1, and cyanidin-3-O-galactoside) formed the most prominent correlation group (highlighted in magenta in Figure 4), with pair-wise correlations ranging from r = 0.71–0.87 (p < 0.001). Epicatechin showed moderate correlations with catechin (r = 0.50, p < 0.001) and procyanidin B1 (r = 0.47, p < 0.001). Since procyanidin B1 is a dimer containing epicatechin and catechin, some correlation among these three compounds is expected. In the cluster analysis, however, epicatechin grouped with luteolin (r = 0.54, p < 0.001) as indicated by the orange-colored box in Figure 4A. A second strong correlation pair was observed between eriodictyol and taxifolin (r = 0.71, p < 0.001), shown in teal in Figure 4A. The flavonols quercetin and isoquercetin exhibited one of the strongest correlation coefficients in the dataset (r = 0.80, p < 0.001); this close association is expected because isoquercetin is a glycosylated form of quercetin and typically follows similar abundance patterns. These two com-pounds appear in the blue cluster of Figure 4.”

Comment 14: The values of R² from lines 418, 424, represents the coefficient of determination and not the correlation coefficient (which is equal with the square root of these values).

Response 14:  Thank you very much for catching this mistake. We should have used the symbol r (lower case r) for the correlation coefficients.  We corrected our mistakes in the manuscript.

Comment 15: Considering that PC3 and PC4 express only 21.6% of the phenolic compounds variability, graphs C and D from Figure S4 can be removed.

Response 15: We decided to keep panels C and D. A contribution of ~22% to overall variability is still worthy of a visual inspection. For example, we noted that the phenolic compound taxifolin, did not contribute much to PC1 and PC2, but has the largest loading factor for PC3. 

Comment 16: The Discussion part (section) can be extended/improved.

Response 16: We expanded the Discussion section and now have two extra subsections entitled “4.4 Selective Phenolic Adjustments under Salinity: Roles of Quercetin, Isoquercetin, and Gallic Acid” and “4.5 Study Limitations and Perspectives”

Comment 17:  In the Conclusions the perspective part is missing; please describe more accurate perspective statements for directing further studies.

Response 17: In addition to the new subsection in the discussion section mentioned above, we also added the following sentences to the Conclusion (p.17, starting line 615):

“Among the responsive compounds, quercetin derivatives show particular promise for tracking localized metabolic adjustments to salinity in mature pistachio orchards. Further work linking these compositional changes to physiological traits and field performance will help clarify their utility as biochemical indicators of salinity exposure and adaptation in long-lived perennial crops such as pistachio.”

 

Reviewer 4 Report

Comments and Suggestions for Authors

Summary

The work analyzes the effect of saline irrigation, at two salinity levels (high and low), on the phenolic composition of the pistachio seed coat (Pistacia vera L.) grown in the Grassland Basin Water District, California. The results show that the phytochemical composition of the seed coat varies mainly as a function of the age of the plants, with a higher polyphenol content in older plants. Regarding the salinity regime, quercetin and its derivative isoquercetin are the only compounds that respond significantly, showing higher concentrations in plants irrigated with high salinity water, particularly in more mature individuals. These results seem to confirm the role of quercetin in pistachio salinity tolerance mechanisms.

General comments

Overall, the manuscript is very interesting, well-written and clear. However, some modifications could help to further improve the quality and scientific impact of the work.

• Clarity of the scientific contribution: it is suggested to highlight more explicitly the contribution that the results obtained make, both in terms of advancing research and potential applications in the cultivation of the species. In particular, this aspect should emerge more strongly in the introduction and conclusions.
• Question for clarification: Did the type of saline irrigation and/or the age of the plants lead to variations in pistachio yield in your experiment?
• Insight into the discussion: the author is invited to delve further into the role of compounds such as quercetin within the seed, highlighting the possible physiological advantages associated with elevated levels of this metabolite. For example, could high levels of quercetin help relieve oxidative stress and therefore promote seed germination in salinity conditions?
• Limitations of the study: It would be appropriate to include a brief reflection on the limits of the work in the discussion. In particular, the fact that the phytochemical composition of the seed coat represents an indirect measure of the metabolic profile of the plant. It may be useful to suggest, as a future perspective, the analysis of the phytochemical profile of other tissues (for example, leaves) to confirm and broaden the observations that emerged in this study.

Author Response

Answer to Reviewers

 

We would like to thank all reviewers for taking the time to read and evaluate our manuscript and for making concrete and insightful comments that helped to improve the quality of our manuscript. The revised manuscript has a Track-Changes option, so that you can see exactly what was changed.  The pdf document is without the Track-Changes option to allow for more unobstructed reading. We use the line numbering and pages of the PDF to describe the location of concrete changes listed below.

Overview on most substantial changes based on all four reviews

 

In revising the manuscript, we made several changes in response to the reviewers' comments. The most important scientific update concerns the statistical analysis. The original version used one-factor Kruskal–Wallis tests, which evaluated irrigation treatment independently of planting year. We replaced this with a two-factor aligned rank transform ANOVA (with comparison to a parametric two-way ANOVA), which more appropriately reflects the factorial structure of the experiment. Under this analysis, irrigation treatment effects could be evaluated while accounting for the influence of planting year. As a result, the salinity responsiveness of quercetin, isoquercetin, and also gallic acid became more apparent; gallic acid had been borderline under the one-factor approach, and the updated analysis resolved its smaller treatment effect more clearly.

We also reorganized the manuscript by separating the combined “Results and Discussion” section into two distinct sections, which required adjusting the order and flow of several passages. The Introduction now includes additional background on ion exclusion, compartmentalization, osmotic adjustment, and the potential roles of boron and selenium in pistachio responses to saline irrigation. The Discussion was expanded with paragraphs addressing the roles of quercetin, isoquercetin, and gallic acid, as well as a new subsection outlining study limitations and directions for future work. Finally, we clarified the study’s contribution in the Introduction and Conclusions and updated several references, including those concerning the Folin–Ciocalteu assay and boron/selenium-mediated stress responses.

Reviewer 4:

Comment 1: Clarity of the scientific contribution: it is suggested to highlight more explicitly the contribution that the results obtained make, both in terms of advancing research and potential applications in the cultivation of the species. In particular, this aspect should emerge more strongly in the introduction and conclusions.

Response 1: We appreciate this recommendation. To strengthen the articulation of our scientific contribution, we expanded both the Introduction and the Conclusions

The following text was added (p.4 near line 143, end of Introduction):

“Although several studies have explored pistachio salt tolerance physiologically, few have examined field-based, long-term transitions from high-saline to low-saline irrigation and their respective impact on phenolic metabolism and antioxidant capacity.”

The following exact text was added to the end of Section 5 Conclusion (p. 17, starting line 615):

“Among the responsive compounds, quercetin derivatives show particular promise for tracking localized metabolic adjustments to salinity in mature pistachio orchards. Further work linking these compositional changes to physiological traits and field performance will help clarify their utility as biochemical indicators of salinity exposure and adaptation in long-lived perennial crops such as pistachio.”

Comment 2: Question for clarification: Did the type of saline irrigation and/or the age of the plants lead to variations in pistachio yield in your experiment?

Response 2: Thank you for raising this question. Yield data were not part of the biochemical analysis and are not included in our dataset. Pistachio nut production is strongly influenced by alternate nut-bearing cycles, making it difficult to associate field-level yield changes with irrigation salinity. Because we could not reliably isolate age or salinity effects on yield from alternate bearing patterns, we did not incorporate yield data into the manuscript.

Comment 3: Insight into the discussion: the author is invited to delve further into the role of compounds such as quercetin within the seed, highlighting the possible physiological advantages associated with elevated levels of this metabolite. For example, could high levels of quercetin help relieve oxidative stress and therefore promote seed germination in salinity conditions?

Response 3: We included a new subsection into our Discussion entitled “4.4 Selective Phenolic Adjustments under Salinity: Roles of Quercetin, Isoquercetin, and Gallic Acid”. One paragraph in this new subsection is dedicated to the seed coat. We were not able to find evidence for a role of quercetin in promoting seed germination.

New text in lines 545-554, page 16:

“In pistachio, the seed coat acts as a protective barrier that limits oxidative and environmental damage to the kernel [31,32]. Within this barrier, flavonols such as quercetin and isoquercetin may help maintain local redox balance during prolonged salinity. Because total antioxidant capacity did not differ between irrigation treatments, the observed enrichment of these flavonols likely reflects a compositional shift toward phenolics that are effective in membrane stabilization, ROS scavenging, or modulation of stress-signaling pathways [39]. This interpretation aligns with studies showing that enhanced flux through the flavonol branch via increased FLS activity supports salinity or combined-stress tolerance in alfalfa, cotton, and cress [55–57]. Pistachio may rely on analogous localized adjustments to preserve seed-coat integrity under saline irrigation.”

Comment 4: Limitations of the study: It would be appropriate to include a brief reflection on the limits of the work in the discussion. In particular, the fact that the phytochemical composition of the seed coat represents an indirect measure of the metabolic profile of the plant. It may be useful to suggest, as a future perspective, the analysis of the phytochemical profile of other tissues (for example, leaves) to confirm and broaden the observations that emerged in this study.

Response 4: Thank you very much for these suggestions. We wrote a new subsection entitled “4.4 Study Limitations and Perspectives” and incorporated the idea of extending future studies into more tissues in the following two sentences:   

“Because only the seed coat was analyzed, potential metabolic adjustments in other tissues—such as leaves, hulls, or roots—remain unresolved. The dataset therefore provides a biochemical snapshot of a long-term field system rather than a temporal profile of salinity adaptation.”

And:

“Multi-year sampling that couples ion analysis with tissue-wide metabolite profiling will be needed to link phenolic composition more directly to physiological and agronomic outcomes.”

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the author's sincere responses, particularly that they have addressed each of my concerns and revised and supplemented the revision based on suggestions.

The revision now have adjusted the title, and expanded the introduction and discussion sections—which has rendered the presentation of the research data more rigorous. 

While I remain cautious about some of the responses, this study comprehensively evaluated polyphenolic composition and antioxidant capacity in pistachio seed coats across different ages under saline irrigation conditions based on multi-year survey data. These findings contribute to advancing scientific research in this field, and I therefore agree to accept this manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear editor and authors. 

I am grateful to the authors for considering my comments. The original manuscript has been notably improved in this revised version, and my questions adequately addressed. Please, note that the color of the circles in graphs H, I and J of Figure 3 have to be corrected according to the remaining graphs.

Reviewer 3 Report

Comments and Suggestions for Authors

Given that the authors have considered (most of) the received comments/suggestions, the new version of the manuscript is significantly improved. As such, I appreciate that the article meets the requirements of the journal and can be accepted in present form.