Phenolic Content and Phenolic Acid Composition of Einkorn and Emmer Ancient Wheat Cultivars—Investigation of the Effects of Various Factors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. One part of the study statistically compares the effect of studied genotypes (cultivars/varieties), crop year, two growing sites and meteorological conditions on the phenolic and phenolic acid content. The other part of the study is a statistical comparison of the effect of ancient wheat genotypes, crop year (which is 2018, 2019, 2020, 2021) and all 9 (?) cropping sites on the phenolic content and phenolic acid content. That should be highlighted somewhere in the introduction section. The title should also note that this research studies the effects of different factors on the phenolic content and phenolic acid content.
2. Somewhere in the introduction section should also be highlighted that in the literature, a lack of comprehensive studies and meta-analyses data concerning phenolic and phenolic acid content of ancient wheat genotypes exists. Based on this scientific lack of data, the present study was conducted. My suggestion is that lines 680-685 from the conclusion section be inserted in the introduction section.
3. The quality of the manuscript would be better if lines 97-106 were replaced in the discussion section.
4. The scientific names of the species should be in italics.
5. It should be specified in all text sections what the studied genotypes (cultivars, varieties or cultivars and varieties) are. There’s a difference between cultivar and variety. This should also be noted in the title. Probably “cultivars” could be replaced with “genotypes”.
6. The tables should have a short explanatory title and caption. Any special characters or icons, special abbreviations need to have a corresponding explanation in the caption.

-Table 1 should have a short explanatory caption with a smaller font. Part of the caption below Table 1 should be inserted into the main text (lines 131-136). The sentence in line 136, “Codes of the…” could be included in the caption below Table 1. There are abbreviations in this table that are not defined.

-Same suggestion for tables 2 and 3. Table 2 should have a short explanatory title and a caption. The title of Table 2 is “Effect of wheat cultivars and growing years on phenolic concentrations (µg FAE g−1 DM) of ancient wheat in Martonvásár (conventional growing conditions) in 2019-2020.” The sentence in lines 317-319 should be inserted below Table 2 with a smaller font.

- Table 3 should have a short explanatory title and a caption. The title of Table 3 is “Effect of wheat cultivars and growing years on phenolic concentrations (µg FAE g−1 DM) of ancient wheat in Želiezovce (organic growing conditions) in 2019-2020.”  The sentence in lines 336-338 should be inserted below Table 3 with a smaller font.

-Same suggestion for table 8. The title of Table 8 is “The result of Analysis of Variance type I on the phenolic compound, total phenolic acids, and seven types of phenolic acid against wheat cultivar and year at site Martonvásár and Želiezovce.” The sentence in lines 601-602 should be inserted below Table 8.

-Same suggestion for Table 9.

7. The figures/tables should be inserted into the main text close to their first citation. My suggestion when inserting the figure/table in the text is to follow the order: first, mention the figure/table in the text, then the concrete figure/table and then the description of the results presented on this figure/table.
8. The quality of the manuscript would be improved if the used abbreviations were defined the first time they appear in each of the three sections: the abstract, the main text, and the first figure or table. When defined for the first time, the abbreviation should be added in parentheses after the written-out form. Once the abbreviated name is included in the text, there’s no need to write it, and you could use its abbreviation.
9. It should be clarified in the text and the tables how long the studied period (two or three years) is and which years are studied (2018 and 2019; 2019 and 2020, or seasons of 2019/2020 and 2020/2021).
10. It would be better if, in the Materials and Methods section, a subsection on the studied period (years/seasons) were included. Somewhere in this section should be mentioned that some of the meteorological conditions as temperature, precipitation and etc., are measured for the studied periods.

Author Response

Reviewer #1 

Comments 1: One part of the study statistically compares the effect of studied genotypes (cultivars/varieties), crop year, two growing sites and meteorological conditions on the phenolic and phenolic acid content. The other part of the study is a statistical comparison of the effect of ancient wheat genotypes, crop year (which is 2018, 2019, 2020, 2021) and all 9 (?) cropping sites on the phenolic content and phenolic acid content. That should be highlighted somewhere in the introduction section. The title should also note that this research studies the effects of different factors on the phenolic content and phenolic acid content. 

Response 1: Thank you for highlighting this important distinction.  We have included the suggested ideas in lines 87-91 of the introduction section. We have also retitled and subtitled the article “Phenolic Content and Phenolic Acid Composition of Einkorn and Emmer Ancient Wheat Cultivars - Investigation of the Effects of Various Factors”. 

Comments 2: Somewhere in the introduction section should also be highlighted that in the literature, a lack of comprehensive studies and meta-analyses data concerning phenolic and phenolic acid content of ancient wheat genotypes exists. Based on this scientific lack of data, the present study was conducted. My suggestion is that lines 680-685 from the conclusion section be inserted in the introduction section. 

Response 2: We agree with your observation and have incorporated this important context from the conclusion section into the introduction (lines 94–97). This addition strengthens the rationale for our study.

Comments 3: The quality of the manuscript would be better if lines 97-106 were replaced in the discussion section. 

Response 3: We have acted upon your valuable suggestion and relocated this content to the discussion section (now appearing in lines 448–464). We have also slightly rephrased this paragraph. 

Comments 4: The scientific names of the species should be in italics. 

Response 4: We have modified the scientific names of species to italics. 

Comments 5: It should be specified in all text sections what the studied genotypes (cultivars, varieties or cultivars and varieties) are. There’s a difference between cultivar and variety. This should also be noted in the title. Probably “cultivars” could be replaced with “genotypes”. 

Response 5: We appreciate your careful attention to terminology. We chose the expression ’cultivar’ as it combines both the homogeneous varieties or variety candidates (genetically identical) and the landraces (consisting of a combination of genotypes, with a low level of genetic diversity existing within) to be used when referring to any category of these.  We included an explanatory part to this in the 2.1 section of Materials and Methods. In Table 1, we have changed the title heading to ’cultivar’ while each cultivar is explained further in the table, to which category it belongs: registered variety, variety candidate, or landrace. 

Comments 6: The tables should have a short explanatory title and caption. Any special characters or icons, special abbreviations need to have a corresponding explanation in the caption. 

-Table 1 should have a short explanatory caption with a smaller font. Part of the caption below Table 1 should be inserted into the main text (lines 131-136). The sentence in line 136, “Codes of the…” could be included in the caption below Table 1. There are abbreviations in this table that are not defined. 

-Same suggestion for tables 2 and 3. Table 2 should have a short explanatory title and a caption. The title of Table 2 is “Effect of wheat cultivars and growing years on phenolic concentrations (µg FAE g−1 DM) of ancient wheat in Martonvásár (conventional growing conditions) in 2019-2020.” The sentence in lines 317-319 should be inserted below Table 2 with a smaller font. 

- Table 3 should have a short explanatory title and a caption. The title of Table 3 is “Effect of wheat cultivars and growing years on phenolic concentrations (µg FAE g−1 DM) of ancient wheat in Želiezovce (organic growing conditions) in 2019-2020.”  The sentence in lines 336-338 should be inserted below Table 3 with a smaller font. 

-Same suggestion for table 8. The title of Table 8 is “The result of Analysis of Variance type I on the phenolic compound, total phenolic acids, and seven types of phenolic acid against wheat cultivar and year at site Martonvásár and Želiezovce.” The sentence in lines 601-602 should be inserted below Table 8. 

-Same suggestion for Table 9. 

Response 6: Thank you for your suggestion. Accordingly, we have extended the title of the tables by indicating the year of the experiments, and the captions have been explained. 

- The following explanatory title has been given to Table 1. “The analysed emmer and einkorn cultivars according to their cultivar type, country of origin, and their cultivation sites between 2019 and 2020”. Notes below the table (lines 131-137) have been inserted in the main text (lines 114-121), and the explanation of the codes has been retained in the small text below the table. One lettering was incorrect in this table because a space was missing (BZ, which is Bugac (B) and Zalaszentlászló (Z)), but it has been corrected. 

- The title of Table 2 has been modified to “Influence of wheat cultivars and cropping years on the concentration of phenolic compounds (µg FAE g⁻¹ DM) of ancient wheat grown under conventional conditions in Martonvásár in 2019-2020". As suggested, the second half of the title has been inserted as a small-italics legend below the table. 

- The title of Table 3 has been modified to “Influence of ancient wheat cultivars and cropping years on the concentration of phenolic compounds (µg FAE g ⁻¹ DM) of ancient wheat grown under organic conditions in Želiezovce in 2019-2020". As recommended, the second half of the title has been inserted as a small-italics legend below the table. 

- Table 8 has modified (according to the other reviewer’s suggestion) into Table 6 and its title has been modified to “The result of Analysis of Variance type I on the phenolic compound, total phenolic acids, and seven types of phenolic acid against wheat cultivar and year at site Martonvásár and Želiezovce (2019, 2020)”, so that the first part of the title was kept and the second part was placed below the table as a note text with smaller font. 

- Table 9 has changed (according to the other reviewer’s suggestion) into Table 7 “The result of analysis of variance type III on the phenolic compound, total phenolic acid, and individual phenolic acid contents against wheat cultivar, site, and year (2019, 2020)“. The first part of the title was kept and the second part was placed below the table as an explanatory note with smaller font. 

Comments 7: The figures/tables should be inserted into the main text close to their first citation. My suggestion when inserting the figure/table in the text is to follow the order: first, mention the figure/table in the text, then the concrete figure/table and then the description of the results presented on this figure/table. 

Response 7: We have repositioned all figures and tables closer to their initial references in the text.

Comments 8: The quality of the manuscript would be improved if the used abbreviations were defined the first time they appear in each of the three sections: the abstract, the main text, and the first figure or table. When defined for the first time, the abbreviation should be added in parentheses after the written-out form. Once the abbreviated name is included in the text, there’s no need to write it, and you could use its abbreviation. 

Response 8: This suggestion has been fully implemented. We have replaced the specific terms in the text and in the abstract with their corresponding abbreviations. As it is important for tables to be readable out of context (e.g., for use in PPT presentations), we have chosen not to use abbreviations in several cases (e.g., phenolic compounds are not abbreviated to PCs).

Comments 9: It should be clarified in the text and the tables how long the studied period (two or three years) is and which years are studied (2018 and 2019; 2019 and 2020, or seasons of 2019/2020 and 2020/2021). 

Response 9: Thank you for pointing this out. We have clarified the specific years of study in all relevant table and figure titles and throughout the manuscript text for consistency and clarity.

Comments 10: It would be better if, in the Materials and Methods section, a subsection on the studied period (years/seasons) were included. Somewhere in this section should be mentioned that some of the meteorological conditions as temperature, precipitation and etc., are measured for the studied periods. 

Response 10: A new subsection (2.2.2) has been added to the Materials and Methods section detailing the meteorological conditions during the study period. Figures A2 a–d in the appendix visually support this information.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Authors are requested to revise the manuscript in light of the following suggestions-

1.The abstract is overly detailed, lengthy and dense. You include specific cultivar codes (e.g., GT-1669), percentages, and detailed acid rankings, which might be too specific for this section. You can summarize major trends rather than list precise data.

2. 
   In the “Introduction” section, some paragraphs are too long and packed with information (e.g., lines 35–74). You can break them. Some citations are overly clustered (e.g., [14–18] and [23–26]). Please summarize or select the most representative ones.

3.Line 69 refers to references "[23–2626]", which appears to be a formatting or typographical error. This should be corrected.

4.In “2.1. Plant Materials and Experimental Design” section, the plot size and replication for on-station trials are well defined (1×50 m plots in 4 reps), but the on-farm trial description is vague in terms of replicates, randomization, or control measures. Please clarify whether any randomization or standardization was attempted across on-farm sites, and how data variability from these trials was managed statistically.
Only two modern wheat controls were included. It's unclear why these specific varieties were chosen and whether they represent average or high-performing genotypes.
Please justify the choice of control wheat varieties in terms of their phenolic content or agronomic performance.

5.Figure 1 is referenced but not described in terms of what each location represents in the context of einkorn vs. emmer distribution. Please add a more detailed caption or table to clarify which cultivars were planted where (perhaps as supplementary material).

6.In “2.2.1On-station and on-farm trials” section the number of replications and cultivar-site combinations varied significantly, which might introduce bias or reduce statistical power. A clearer justification or strategy for managing unbalanced data would be helpful. Although it's mentioned that farmers followed their usual routines, the lack of detail on specific agronomic practices (e.g., seeding rates, weed control, irrigation) could hinder reproducibility and interpretation of cultivar performance.

7. “2.3. Total, Free, Bound Phenolics and Phenolic Acids Determination” section lacks discussion or validation of extraction recovery or efficiency for both free and bound phenolics. This would be critical for method validation, especially since multiple extraction and hydrolysis steps are involved.

8.Since phenolics can be sensitive to pH, consider noting the pH during extraction (or confirming if it was not controlled) in “2.3.1. Extraction procedure for Free and Bound Phenolics” section. In addition, in this section, the protocol uses alkaline hydrolysis (NaOH), followed by acidification and diethyl ether extraction. It might be useful to reference how effective this method is at releasing cell-wall-bound phenolics in wheat, or whether any alternatives (e.g., enzymatic hydrolysis) were considered. Although the extracts were evaporated and reconstituted, the method of drying (e.g., under nitrogen stream or rotary evaporation) could be made more explicit for clarity.

9. In “3.2. Total, free and bound phenolic acids (TPAs, FPAs, and BPAs)” section, the heavy listing of exact numeric values (e.g., μg/g DM for each cultivar) disrupts the flow. Please summarize these data in tables/graphs only and highlighting trends in text for better readability. The differences are well described, however, there's limited discussion on why certain cultivars (e.g., GT-1669) had higher phenolic acid levels. Please add a discussion on this. Similarly, the nutritional significance of differences in TPAs, FPAs, and BPAs is not well-discussed. For example, is the observed increase in TPA in emmer substantial enough to affect dietary antioxidant intake?
10. Fig. 4 and Fig. 5 should be improved, such as color coding by wheat type (einkorn, emmer, bread wheat) and annotated statistical significance directly on bar plots for visual ease.
11. In “3.3. Individual Phenolic Acids” section, most of the numeric data in the paragraph text is also present in the accompanying tables. Please summarize the trends into tables for specifics to avoid repetition and improve readability.
12. In “3.5. Trends in the phenolic compound and phenolic acid content of all species at all sites” section, the data coverage is impressive, the section is extremely data-heavy and sometimes lacks concise synthesis. Please add a small paragraph at the end of the section summarizing key patterns (e.g., "Overall, emmer cultivars performed better across all environments, with cultivar GT-1669 consistently ranking highest in phenolic content").
13. In “3.5. Trends in the phenolic compound and phenolic acid content of all species at all sites” section, you can add figures or summary heatmaps comparing mean total phenolic and phenolic acid content across species and sites.

 

Author Response

Reviewer #2 

Comments 1: The abstract is overly detailed, lengthy and dense. You include specific cultivar codes (e.g., GT-1669), percentages, and detailed acid rankings, which might be too specific for this section. You can summarize major trends rather than list precise data. 

Response 1: Thank you for this suggestion. We have shortened and simplified the abstract by removing overly detailed content, such as cultivar codes and exact values. At the request of the other reviewer, I have also included abbreviations in the summary. 

 

Comments 2: In the “Introduction” section, some paragraphs are too long and packed with information (e.g., lines 35–74). You can break them. Some citations are overly clustered (e.g., [14–18] and [23–26]). Please summarize or select the most representative ones. 

Response 2: We appreciate your feedback and have broken down the longer paragraphs in lines 35–75 for better flow, and left only the most relevant references in the two cases you mention. 

 

Comments 3: Line 69 refers to references "[23–2626]", which appears to be a formatting or typographical error. This should be corrected. 

Response 3: This was a typo, the duplication has been deleted. 

 

Comments 4.In “2.1. Plant Materials and Experimental Design” section, the plot size and replication for on-station trials are well defined (1×50 m plots in 4 reps), but the on-farm trial description is vague in terms of replicates, randomization, or control measures. Please clarify whether any randomization or standardization was attempted across on-farm sites, and how data variability from these trials was managed statistically.  

Only two modern wheat controls were included. It's unclear why these specific varieties were chosen and whether they represent average or high-performing genotypes. 
Please justify the choice of control wheat varieties in terms of their phenolic content or agronomic performance. 

Response 4: Thank you for pointing this out. In the on-farm experiments, there was only one plot for all cultivars in all sites. Ancient wheat grains collected from each plot were representative composite samples, with only one sample representing each plot. In the case of the on-station experiment in Martonvásár, control wheats, einkorns and emmers samples from four plot replicates were also mixed into one composite sample for each cultivar. This way, we reduce the plot variability since we are more interested in the year, site, and cultivar effect. Next, in the laboratory, four technical replicates from each cultivar sample were made, so in the end, we have four replicate data sets for further analysis. We explain this better now in the manuscript (lines 204-211):  

“Representative composite grain samples were collected from individual plot harvests at different trial locations. Where plot-specific harvesting was not feasible due to technical limitations (such as in Želiezovce), samples were instead obtained at full maturity from three 1 m² randomly selected subplots within each cultivar’s area. In the case of wheat controls, einkorns and emmers in Martonvásár, the samples from four plot replicates were also mixed into one composite sample for each cultivar. This way, we reduce the plot variability since we are more interested in the year, site, and cultivar effect. Next, each sample was divided into four technical replicates from each cultivar for further analysis. These were then analyzed for quality at the Slovak University of Agriculture in Nitra, Institute of Agronomic Sciences, following the outlined procedures”. 
Regarding the winter wheat controls, the selection of these specific two varieties was based on their overall agronomic and quality performance in organic fields. Several years of on-station trials carried out formerly in the organic field of HUN-REN Centre for Agricultural Research (Martonvásár, Hungary) have led to the selection of the most stable yielding variety (Mv Káplár) and the most stable quality variety (Mv Uncia), which latter had also higher than average grain yield. The greatest difference between these two varieties lies in their quality profile. The record yielding Mv Káplár is a soft wheat with low protein content and poor technological quality (mainly useful for biscuits), while Mv Uncia has high bread making quality with good protein and gluten content and outstanding technological quality properties (e.g. extenzograph, alveograph). Both varieties are resistant against most of the wheat diseases (e.g. leaf rust, yellow rust, powdery mildew), therefore they are excellent choice for organic farmers (depending on their processing target: bread or biscuit). Phenolic content of them was not studied formerly, thus these two varieties with largely contrasting quality profile were chosen for this experiment to be able to compare ancient wheats with different types of modern wheats under organic growing conditions. 

 

Comments 5: Figure 1 is referenced but not described in terms of what each location represents in the context of einkorn vs. emmer distribution. Please add a more detailed caption or table to clarify which cultivars were planted where (perhaps as supplementary material). 

Response 5: You are absolutely right. Indeed, we did not include the details of which cultivars were planted at each site in Figure 1 for simplicity. However, we included the details of each wheat cultivar used in the experiments with the sites planted in Table 1 and added Appendix A, Figure A1, for more details. We mentioned this in the caption of Figure 1:  

Figure 1. Map of the study sites (green points) located in Hungary and Slovakia. The two sites indicated in bold (Martonvásár and Želiezovce) were selected for more detailed analysis (see Table 1 & Appendix A for cultivars planted on each site and section 2.3.4 for the selected sites analysis).  

Appendix Figure A1 

The list of 19 wheat and ancient wheat cultivars used in the research  

The grey shade indicates the sites and years of the grown cultivars. 

 

Comments 6: In “2.2.1On-station and on-farm trials” section the number of replications and cultivar-site combinations varied significantly, which might introduce bias or reduce statistical power. A clearer justification or strategy for managing unbalanced data would be helpful. Although it's mentioned that farmers followed their usual routines, the lack of detail on specific agronomic practices (e.g., seeding rates, weed control, irrigation) could hinder reproducibility and interpretation of cultivar performance. 

Response 6: Indeed, you were right. The unbalanced data can introduce a bias in the data interpretation. In this manuscript, we decided to discuss more in-depth the wheat cultivars present on the same sites at two years, i.e., 12 cultivars from Martonvásár and 4 cultivars from Želiezovce as presented in sections 3.1 to 3.4.  

On the other hand, to deal with the overall unbalanced datasets, we carefully look into the descriptive statistics of the data, conducting data exploration to visualize and inspect patterns in the data and conducting Anova type III (see section 3.6 Table 9 for results). Anova type III can handle unbalanced or missing data and explanatory variables that are not independent since it works by using the adjusted sum of squares (see Hector et al., 2010; Smith and Cribble, 2014). The common Anova (type I) used the sum of squares sequentially, while type III calculates the sum of squares from multiple sequential analyses. Every effect in type III is adjusted for all other effects, and the least squares estimates of the value replace the missing data. This way, the sum of squares from type III will be similar to the ones of type I. By combining the visual data interpretation and statistical analysis justification, we are more confident in presenting the results. We mentioned our strategy for analyzing the unbalanced data using Anova type III in section 2.3.4 (lines 284-289): 

“Since not all cultivars were sown at all sites and in both years, cultivars that were grown at least in more than one site or more than one year of observation were subjected to ANOVA Type III, which can deal with unbalanced data (Hector et al., 2010; Smith and Cribble, 2014). This type of ANOVA works by adjusting every effect for all other effects, and the least squares estimates of the value replace the missing data. This way, the sum of squares from type III will be similar to the ones of type I.” 

References:  

Hector, A., Von Felten, S., & Schmid, B. (2010). Analysis of variance with unbalanced data: an update for ecology & evolution. Journal of animal ecology, 79(2), 308-316. 

Smith, C. E., & Cribbie, R. (2014). Factorial ANOVA with unbalanced data: a fresh look at the types of sums of squares. Journal of Data Science, 12(3), 385-403. 

 

Comments 7: “2.3. Total, Free, Bound Phenolics and Phenolic Acids Determination” section lacks discussion or validation of extraction recovery or efficiency for both free and bound phenolics. This would be critical for method validation, especially since multiple extraction and hydrolysis steps are involved. 

Response 7: Thank you for this valuable suggestion. In the present study, we applied well-established extraction procedures for free and bound phenolics as described in the cited literature. We acknowledge that method validation, including recovery assessment, is essential for ensuring the reliability of the data. While we did not perform a full validation of extraction recovery within these analyses for all individual phenolic compounds, the methods were applied consistently across all samples, and replicate analysis showed good reproducibility. We acknowledge the importance of method validation and further confirmation of the extraction efficiency for both free and bound phenolics. 

 

Comments 8: Since phenolics can be sensitive to pH, consider noting the pH during extraction (or confirming if it was not controlled) in “2.3.1. Extraction procedure for Free and Bound Phenolics” section. In addition, in this section, the protocol uses alkaline hydrolysis (NaOH), followed by acidification and diethyl ether extraction. It might be useful to reference how effective this method is at releasing cell-wall-bound phenolics in wheat, or whether any alternatives (e.g., enzymatic hydrolysis) were considered. Although the extracts were evaporated and reconstituted, the method of drying (e.g., under nitrogen stream or rotary evaporation) could be made more explicit for clarity. 

Response 8: Further we appreciate the reviewer’s insightful suggestions regarding:   pH Control: We acknowledge that phenolics are sensitive to pH changes. During the extraction of free phenolics, pH was not strictly adjusted but remained near-neutral as no strong acids or bases were added. In the case of bound phenolics, the alkaline hydrolysis was carried out which provided a basic environment (approximate pH ~13). After hydrolysis, the mixture was acidified to pH ~2 using concentrated HCl before diethyl ether extraction. We agree that referencing the effectiveness of alkaline hydrolysis for releasing cell-wall-bound phenolics in wheat may add important context.  Alkaline hydrolysis is a widely accepted and effective method for releasing ester- and ether-linked phenolics from cereal matrices such as wheat. Although enzymatic hydrolysis has been explored in other studies, we did not employ it in this work due to its longer processing times and potential variability. However, we acknowledge enzymatic methods as a promising alternative. Drying Method: We thank the reviewer for pointing out the need to clarify the drying step. The extracts were evaporated using a rotary evaporator at temperatures to avoid thermal degradation of phenolic compounds. 

 

Comments 9: In “3.2. Total, free and bound phenolic acids (TPAs, FPAs, and BPAs)” section, the heavy listing of exact numeric values (e.g., μg/g DM for each cultivar) disrupts the flow. Please summarize these data in tables/graphs only and highlighting trends in text for better readability. The differences are well described, however, there's limited discussion on why certain cultivars (e.g., GT-1669) had higher phenolic acid levels. Please add a discussion on this. Similarly, the nutritional significance of differences in TPAs, FPAs, and BPAs is not well-discussed. For example, is the observed increase in TPA in emmer substantial enough to affect dietary antioxidant intake? 

Response 9: For better readability, we have deleted the detailed figures that are already included in the tables or graphs and only the trends have been left in the text. The reason why GT-1699 contains the highest PA concentrations is described in lines 464-477 in the following way: “In our study, the emmer cultivar GT-1669 showed total phenolic acid (TPA) concentrations in the highest range among the lines tested. This emmer cultivar, commonly known by its original name 'Schwarzer Eschikon', is a blackish-flaked spelt landscape variety. Its characteristically higher TP content and TPA, BPA and FPA content can be explained by its genetically determined high content of TP compounds containing colouring agents that give the straw a dark purple-blackish colour. It is established that darker cereal phenotypes are frequently associated with higher TP content, likely due to the greater synthesis and accumulation of these bioactive compounds [14, 45, 51]. On the basis of contemporary scientific knowledge and dietary context, the observed elevation in TPA in GT-1669 is nutritionally meaningful: regular consumption of products made from this emmer can substantially boost PA, and thus, dietary antioxidant intake compared to typical cereal-based foods. ” 

 

Comments 10: Fig. 4 and Fig. 5 should be improved, such as color coding by wheat type (einkorn, emmer, bread wheat) and annotated statistical significance directly on bar plots for visual ease. 

Response 10: Thank you for pointing this out. We have revised both figures (Figs. 4 and 5) per request and the corresponding Figure 2and 3. The different wheat type is now annotated with different colors and significance between years, which is integrated into the graph. We also use different color coding for phenolic compounds and phenolic acid datasets. In addition, the figure captions were revised to add more clarity. 

Figure 2. The effect of growing year on mean ± SD total phenolic compound of selected cereal cultivars at site Martonvásár (2019, 2020). Note that the two common wheat varieties were only present in one year (Mv Káplár in 2019 and Mv Uncia in 2020); therefore, they were not tested for year effect but were shown as a comparison. The significance (Anova p-value) differences between the two years of measurements are annotated in each cultivar, with significant values appearing in bold. 

Figure 3. The effect of growing year on mean ± SD total phenolic compound of selected wheat cultivars at site Želiezovce (2019, 2020). The significance (Anova p-value) differences between the two years of measurements are annotated in each cultivar, with significant values appearing in bold.

Figure 4. Effect of cropping year on mean ± SD of total phenolic acids in selected wheat cultivars at site Martonvásár (2019, 2020). Note that the two common wheat varieties were only present in one year (Mv Káplár in 2019 and Mv Uncia in 2020); therefore, they were not tested for year effect but were shown as a comparison. The significance (Anova p-value) differences between the two years of measurements are annotated in each cultivar, with significant values appearing in bold.

Figure 5. The effect of growing year on mean ± SD total phenolic acids of selected wheat cultivars at site Želiezovce. The significance (Anova p-value) differences between the two years of measurements are annotated in each cultivar, with significant values appearing in bold. 

 

Comments 11: In “3.3. Individual Phenolic Acids” section, most of the numeric data in the paragraph text is also present in the accompanying tables. Please summarize the trends into tables for specifics to avoid repetition and improve readability. 

Response 11: Thank you for your suggestion. We have changed the 4 tables (Table 4-7) containing free and bound individual phenolic acids into 2 tables (Table 4 and 5) with the total value instead. We also revised the text in this section to make it more concise and easier to read: 

“An interesting aspect of our research is that the individual PA concentrations of total, free, and bound fractions were analyzed. Even though the total individual PAs concentrations differ between cultivars (Table 4 and 5), their percent share showed a similar trend. Ferulic acid accounts for 77.2-78.9% of the total individual PAs measured, followed by p-coumaric (6.1-7.0%), sinapic acid (4.8-5.3%), salicylic acid (3.0-3.5%), p-HBA (2.5-2.7%), caffeic acid (2.2-2.5%), and syringic acid (2.1-2.4%). The predominance of ferulic acid was also reported in other scientific publications [26, 33, 54].

Table 4 Effect of different wheat and ancient wheat cultivars and growing years on the total individual phenolic acid concentration (µg g⁻¹ DM) of samples originating from Martonvásár (2019, 2020)

Species, Cultivars	Ferulic Acid	p-HBA 1	p-Coumaric Acid	Syringic Acid	Sinapic Acid	Salycylic Acid	Caffeic Acid
T. aestivum
Mv Káplár	358.8 ± 20 a	11.8 ± 0.2 a	28.5 ± 0.7 a	10.9 ± 0.5 a	21.7 ± 0.5 a	14 ± 0.6 a	10.1 ± 0.4 b
Mv Uncia	322 ± 31.6 b	10.5 ± 0.2 b	29 ± 0.5 a	9.4 ± 0.2 b	22.1 ± 0.8 a	14.6 ± 0.3 a	9.3 ± 0.4 cd
T. monococcum
Mv Alkor St	208.6 ± 149.7 d	6.9 ± 4.7 g	17.3 ± 11.8 e	5.9 ± 4.2 g	13.6 ± 9.7 de	8.6 ± 5.9 f	6.3 ± 4.3 gh
Mv Esztena	176.3 ± 123.7 e	6 ± 4 h	13.8 ± 9.4 g	5.2 ± 3.5 h	11.1 ± 7.3 g	7.4 ± 4.9 g	5.6 ± 3.8 i
Mv Menket	235.6 ± 64.8 c	7.4 ± 2 f	20.3 ± 5.4 d	6.7 ± 1.6 f	14.4 ± 2.8 d	9.6 ± 2.8 e	6.7 ± 1.8 g
T. dicoccum
GT-1399	303.1 ± 50.4 b	9.7 ± 1.7 d	24.8 ± 3.8 bc	8.7 ± 1.3 cd	18.9 ± 3.4 b	12.2 ± 1.8 c	9.4 ± 1.6 c
GT-1400	199.7 ± 23.1 d	6.8 ± 0.8 g	15.8 ± 1.2 f	5.6 ± 0.5 g	12.4 ± 1.1 f	8.1 ± 0.7 f	6.1 ± 0.4 hi
GT-143	200.1 ± 8.7 d	6.8 ± 0.3 g	16.9 ± 0.7 ef	5.7 ± 0.2 g	12.8 ± 0.5 bf	8.3 ± 0.6 f	5.7 ± 0.3 i
GT-1669	365 ± 13.3 a	11.8 ± 0.5 a	29.1 ± 1.2 a	10.5 ± 0.3 a	22.1 ± 0.8 a	14.1 ± 1 a	10.9 ± 0.7 a
GT-1971	295.9 ± 141.5 b	9.9 ± 4.5 bcd	25.1 ± 11.8 cd	8.8 ± 4.1 cd	18.4 ± 7.4 b	13 ± 5.9 b	9 ± 3.8 cd
GT-381	298.2 ± 32.8 b	9.7 ± 0.8 cd	23.6 ± 1.3 d	8.5 ± 1.1 d	18 ± 2.8 b	11.9 ± 0.8 c	8.4 ± 0.6 e
GT-831	200.5 ± 22.1 d	6.5 ± 0.9 g	16.5 ± 2.7 g	5.7 ± 1 g	12.3 ± 1.9 f	8.3 ± 1.4 f	6 ± 0.6 hi
Holland emmer	252.9 ± 15.7 c	8.2 ± 0.2 e	20.4 ± 0.8 e	7.6 ± 0.2 e	15.9 ± 0.8 c	10.4 ± 0.7 d	7.5 ± 0.2 f
Mv Hegyes St	300.9 ± 125 b	10.2 ± 4.4 bc	24.7 ± 10.1 bc	9 ± 3.6 bc	18.8 ± 8.7 b	13.1 ± 6.3 b	8.8 ± 3.4 de
p cultivars (C)	***	***	***	***	***	***	***
2019	308.9 ± 73.6 a	10.2 ± 2.4 a	25.3 ± 6.1 a	8.9 ± 2.2 a	19 ± 4.6 a	12.9 ± 3.2 a	9.2 ± 2.2 a
2020	210.6 ± 90.4 b	6.9 ± 2.8 b	17.3 ± 7.1 b	6.1 ± 2.6 b	13.4 ± 5.6 b	8.5 ± 3.5 b	6.2 ± 2.4 b
p year (Y)	***	***	***	***	***	***	***
p C × Y	***	***	***	***	***	***	***

¹p-hydroxybenzoicacid. All values are expressed as means ± standard deviations (SD), based on four replicates and data collected over two consecutive years (n=14 for varieties, n=28 for years). Mean values within columns that share the same letter are not significantly different at the 5% significance level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) test following one-way ANOVA. Statistical significance is denoted as ***p<0.001, **p<0.05.

 

Table 5 Effect of ancient wheat cultivars and cropping years on total individual phenolic acid concentration (µg g⁻¹ DM) of samples originating from Želiezovce (2019, 2020)

Species, Cultivars	Ferulic Acid	p-HBA 1	p-Coumaric Acid	Syringic Acid	Sinapic Acid	Salicylic Acid	Caffeic Acid
T. monococcum
Mv Alkor St	105.3 ± 5.8 c	3.3 ± 0.1 c	8.5 ± 0.3 c	3.1 ± 0.1 c	6.6 ± 0.2 c	4.1 ± 0.1 c	3.2 ± 0.1 b
T. dicoccum
GT-1400	177.1 ± 10.8 a	5.9 ± 0.5 a	15.1 ± 1.3 a	5.4 ± 0.2 a	11.9 ± 0.8 a	7.3 ± 0.5 a	5.1 ± 0.3 a
GT-831	164.8 ± 33.6 b	5.7 ± 1.1 b	13.8 ± 2.4 b	4.9 ± 0.9 b	10.1 ± 2.0 b	6.9 ± 1.3 b	5.1 ± 1 a
Mv Hegyes	92.0 ± 5.0 d	3.0 ± 0.1 d	7.6 ± 0.3 d	2.7 ± 0.1 d	5.9 ± 0.3 d	3.6 ± 0.1 d	2.7 ± 0.1 c
p cultivars (C)	***	***	***	***	***	***	***
2019	139.2 ± 46.2 a	4.6 ± 1.6 a	11.5 ± 3.8 a	4.2 ± 1.4 a	8.8 ± 2.8 a	5.6 ± 2.0 a	4.2 ± 1.4 a
2020	130.4 ± 36.3 b	4.3 ± 1.4 b	11 ± 3.4 b	3.8 ± 1.2 b	8.4 ± 2.6 b	5.3 ± 1.6 b	3.9 ± 1.0 b
p year (Y)	***		***	***	***	***	***
p C × Y	***		***	***	***	***	***

¹p-hydroxybenzoicacid. All values are expressed as means ± standard deviations (SD), based on four replicates and data collected over two consecutive years (n=4 for varieties, n=8 for years). Mean values within columns that share the same letter are not significantly different at the 5% significance level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) test following one-way ANOVA. Statistical significance is denoted as ***p<0.001, **p<0.05.

The bound form of the seven PAs was always higher than their free form. The share of seven PAs in their bound form is similar to the total share; however, their free form showed a different trend. Free form of ferulic acid accounts for 57.3-59.2%, followed by p-coumaric (14.3-16%), syringic acid (9.9-11.1%), sinapic acid (6.2-7.1%), p-HBA (4.4-5.1%), salicylic acid (3.0-3.7%), and caffeic acid (1.5-2.1%).

Baranski et al. [3] reported that ferulic acid was dominant among all PCs in wheat, representing over 72% of free PAs and over 95% of bound ones. Lacko-Bartošová et al. [45] reported higher free PAs concentration in emmer than in wheat. Ferulic acid was the predominant free phenolic acid (66.3%) detected across the wheat genotypes, followed by syringic acid (11.7%), sinapic acid (7.4%), p-hydroxybenzoic acid (5.3%), salicylic acid (3.8%), p-coumaric acid (3.6%), and caffeic acid (2.1%). In contrast, Li et al. [35] found a lower rate of FPAs in emmer than in bread wheat. It has been pointed out that there is a lack of data on the presence of FPAs in ancient wheat, but it is also recommended that further studies are carried out with various ancient and modern wheat species and varieties [15]. At the Želiezovce organic site, a similar trend was observed, with ferulic acid being the most dominant aggregate in measured samples (Table 5). ” 

 

Comments 12: In “3.5. Trends in the phenolic compound and phenolic acid content of all species at all sites” section, the data coverage is impressive, the section is extremely data-heavy and sometimes lacks concise synthesis. Please add a small paragraph at the end of the section summarizing key patterns (e.g., "Overall, emmer cultivars performed better across all environments, with cultivar GT-1669 consistently ranking highest in phenolic content"). 

Response 12: We agree with your suggestion. We rewrote the section to make it more concise, and we added the heatmap figures (see the following question) as requested to support the explanation:  

“Nineteen wheat cultivars grown in 2019 and 2020 at different sites showed a wide range of PC and PA content (Figure 6 and 7). The results of samples from other on-farm locations (where only one year of data was available) were not relevant for drawing conclusions but were suitable for observing trends. PC and PA values followed the same trend where their bound form (PC: 82.5-89.0%, PA: 91.9-93.6%) was consistently higher than their free form (PC: 11.0-17.5%, PA: 6.4-8.1%).

In both PC and PA, the highest value was shown by emmer GT-831 grown in Füzesgyarmat (PC: 1254.0 ± 108.9 µg FAE g⁻¹ DM, PA: 748.1 ± 0.02 µg g⁻¹ DM) and the lowest value was shown by einkorn Mv Esztena (PC: 117 ± 2.0 µg FAE g⁻¹ DM, PA: 66.9 ± 3.5 µg g⁻¹ DM). The PC and PA values of emmer GT-831 (as well as others) differed in each site and year, showing the site and year effect. The three sites, i.e., Bugac (2019), Füzesgyarmat (2019), and Martonvásár (2019, 2020), showed a higher PC and PA than the others (Figure 6 and 7). Meanwhile, wheat cultivars grown in 2020 showed a lower PC and PA than in 2019, as demonstrated by sites Martonvásár, Pásztó, and Želiezovce.”

 

Comments 13: In “3.5. Trends in the phenolic compound and phenolic acid content of all species at all sites” section, you can add figures or summary heatmaps comparing mean total phenolic and phenolic acid content across species and sites. 

Response 13: Thank you for the great idea! We have made two heatmaps to summarize and compare the mean total phenolic compound and phenolic acid across 19 wheat cultivars and 10 sites. Since the year effect was also pronounced in two previously mentioned sites (Martonvásár and Želiezovce), we added this to the heatmaps.  

Figure 6. Heatmaps of the mean total phenolic compound of 19 wheat varieties were measured at 10 sites in two years (2019, 2020) of measurements. The darker shade represents a higher mean phenolic compound value. The sites were B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, and Ze: Želiezovce. See Fig. 1 and Table 1 for more details on the sites and the cultivars.

 

Figure 7. Heatmaps of the mean total phenolic acid of 19 wheat varieties were measured at 10 sites in two years (2019, 2020) of measurements. The darker shade represents a higher mean phenolic acid value. The sites were B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, and Ze: Želiezovce. See Fig. 1 and Table 1 for more details on the sites and the cultivars.

Similar to PC and PA results, in all individual phenolic acids measured, the proportion of bound forms was higher than that of free ones (62.4-94.9% vs. 5.1-37.6%). The bound to free PA ratio was the highest in ferulic and caffeic acid (multiple wheat varieties). On the contrary, the lowest bound to free PA ratio was shown by syringic acid (einkorn variety GT-2139 and emmer variety Roter). The overall mean bound to free ratio of each PA, starting from the highest to the lowest, was ferulic acid < caffeic acid < salicylic acid < sinapic acid < p-hydroxybenzoic acid < p-coumaric acid < syringic acid. It seems that the TPA content is positively correlated with the bound to free ratio of PAs, meaning that high PA content is connected to the production of more bound than free PAs.”

 

Author Response File: Author Response.docx

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The manuscript has been sufficiently improved to warrant publication in Agriculture.