Synthetic Auxins Toxicity: Effects on Growth and Fatty Acid Composition in Etiolated and Green Spring Wheat Seedlings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please find the review of the manuscript of the paper entitled: "Synthetic auxins toxicity: effects on growth and fatty acid 2 composition in etiolated and green spring wheat seedlings"

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Dear reviewer,

We sincerely thank you for the thorough evaluation and constructive comments on our manuscript.

We have carefully considered all comment and suggestions and have revised the manuscript accordingly. Please find the detailed responses below and the corresponding revisions/corrections highlighted yellow in the re-submitted files. We have also revised all the figures based on your comments. The new figures are presented in the revised version of the manuscript and in the attached files.

The English version of the manuscript was reviewed and edited by a professional linguist translator. We hope these revisions have strengthened the manuscript, and hope these revisions and responses will meet your requirements.

 

The Abstract should also be described more clearly.

According to the comment, the abstract was described more clearly.

1. Does the introduction provide sufficient background and include all relevant references?
2. a) Lack of key references regarding fatty acids in plant stress

We sincerely thank the reviewer for the important comment. References and corrections regarding fatty acids in plant stress have been added to the text accordingly. Lines 105-112

Membrane structure and fluidity, which determine membrane permeability and the activity of membrane-bound enzymes, largely depend on the content of polyunsaturated fatty acids (PUFAs) in membrane lipids [31]. High PUFA content, particularly linolenic acid (С18:3), is associated with increased plant tolerance to drought, salinity, and low and high temperatures [32,33,34,35,36]. PUFAs also serve as precursors of bioactive molecules, such as phytooxylipins and nitroalkenes, and participate in stress signaling [37,38,39,40].

1. b) Insufficient discussion of previous studies on 2,4-D and CLD in cereals

We sincerely thank the reviewer for the significant comment. References and corrections about previous studies on 2,4-D and CLD in cereals have been added to the text accordingly. Lines 89-96.

Conversely, information regarding the influence of synthetic auxins on the physiology and metabolism of cereals remains scarce and fragmented and contradictory [17, 24,25,26,27]. However, studies have shown that 2,4-D at early developmental stages can cause stem shortening, suppress the development of leaves, roots, and spikelets, and reduce wheat yields [25]. Overlapping CLD treatments in winter wheat also resulted in visible plant damage and a 19% yield reduction [26]. Pyone et al. (2025) found no inhibitory effect of CLD on root and shoot growth in winter wheat [27].

1. c) Weak justification for choosing wheat as a model

We thank the reviewer for the comment. The following corrections and references have been added to the Introduction: Lines 80-86

Triticum aestivum L. is one of the major agricultural crops and the most widely consumed cereal in the world [17]. The adaptability of wheat to environmental conditions has enabled its cultivation across a broad range of temperate climates [18]. Under optimal conditions, wheat yields can exceed 10 t/ha [17]. However, exposure to adverse environmental factors leads to substantial yield reductions [19]. Therefore, it is crucial to investigate all factors that may negatively affect wheat productivity, including the effects of herbicides.

1. d) No reference to earlier studies on the effects of herbicides on lipid composition

We sincerely thank the reviewer for significant comment. References to earlier works have been added to the manuscript and corrections have been made. Lines 112-123

Lipid and fatty acid composition can be altered by herbicides. Aryloxyphenoxypropionates and cyclohexanediones inhibit plastidic acetyl-CoA carbox-ylase; thiocarbamates and chloroacetanilides inhibit elongases and С18:2 desaturases; ac-etamides, chloroacetanilides, oxyacetamides, and tetrazolinone inhibit the synthesis of very long-chain fatty acids (VLCFAs) [41,42,43,44,45]. Research on the effects of auxin-type herbicides on plant lipid metabolism is limited. NAA and 2,4-D enhanced lipid synthesis and accumulation in cells of the unicellular alga Chlorella pyrenoidosa under mixotrophic cultivation [46]. 2,4-D increased membrane permeability, LPO product levels, and reduced the С18:2/С18:1 fatty acid ratio in the fungus Trichoderma harzianum, which is widely used for protecting plants against pathogens and abiotic stresses [47]. No studies examining the effects of CLD on lipid synthesis in plants were found in the available literature.

1. e) Section on fatty acid metabolism (lines 81–89) is too general

We sincerely thank the reviewer for the important comment. References to earlier works have been added to the manuscript and corrections have been made as in response to point 1a).

1. f) Lack of citations for key statements on the toxicity of NAA vs 2,4-D

We thank the reviewer for the valuable comment. The text has been edited and references added. Lines 59-62

«NAA is used in agriculture to prevent fruit drop and improve fruit quality. It stimulates root formation and sugar accumulation [8,9]. There are studies indicating both lower toxicity of NAA [10] and similar toxic effects of NAA and 2,4-D on plants [11]».

2. Is the research design appropriate?
3. a) Two concentrations (1 and 10 μM) enable observation of dose–response relationships

Concentrations of 1 and 10 μM are lying at the edges of the effective concentration range of the studied synthetic auxins. The observed changes in growth, pigment content, and some fatty acid levels were more pronounced for NAA and 2,4-D at higher concentrations. A more detailed description of the preliminary experiments is provided in the answer to point 2b).

Weaknesses / Limitations:

1. b) No explanation is provided for why the concentrations of 1 and 10 μM were chosen

The effective concentrations of synthetic auxins were selected in preliminary experiments. The effects of NAA, 2,4-D, and CLD were analyzed at the following concentrations: 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM. Such a wide range of values for the preliminary experiments was due to the fact that in our experiments with a suspension cell culture of Arabidopsis thaliana L., very high values of 2,4-D and CLD — from 500 μM — caused an impact on culture viability, ROS levels, and the potential on the inner mitochondrial membrane (Fedyaeva et al., 2024, https://doi.org/10.1007/s40502-024-00806-3). However, experiments with intact plants were revealed that concentrations of synthetic auxins (primarily 2,4-D and NAA) exceeding 10 μM caused significant disruption to seedling growth, leading to mass mortality. Therefore, we excluded high concentrations of synthetic auxins. Concentrations below 1 μM had no noticeable effect on seedlings growth. It was decided not to include data from preliminary experiments in this manuscript to avoid overloading the rather lengthy manuscript. As you rightly noted, the section of the manuscript describing the changes observed in fatty acid composition, even when using the two concentrations of the effective range, presents a large array of numerical data. Adding an intermediate concentration would only complicate their presentation and discussion.

1. c) Study does not consider that observed effects may be specific to the wheat cultivar used

Thanks to the reviewer for the valuable comment. The following phrase has been added to the Conclusion. Lines 832-833

As the obtained results may be cultivar-specific, further research on various winter and spring wheat cultivars is required to identify general patterns.

3. Are the methods adequately described?
4. a) Missing information on biological replicates in some experiments

Section 4.8 states that «At least four independent experiments were performed for each experimental variant» (lines 797-798). The number of biological repeats is also indicated in the description of each figure and table.

1. b) Insufficient details on GC-MS calibration

According to the comment, next additions have been made to the text. Lines 788-795.

 

«The relative content of FAs was determined by the internal normalization method and expressed as weight percent (wt%) of the total FA content in the sample, taking into account the FA response factors. Nonadecanoic acid (С_19:0), which is absent in the sample, was used as the internal standard. The absolute content of each fatty acid (P_i) was calculated from the total weight of FA methyl esters (FAME) in the sample (P _{Total FAME}) and the relative percentage of that acid (C_{i % rel}) using the following formula: P_i =P _{Total FAME}*C_{i wt%} / 100. The content of FAs was expressed as μg Í g^–1 FW.»

 

1. c) No information on sample storage prior to analysis

Thank you very much for your valuable comment. All physiological and biochemical parameters were analyzed using freshly collected samples. The corresponding phrases has been added to sections 4.1, 4.3, 4.4, 4.5, and 4.6.

1. d) Electrolyte leakage method (lines 631–642) – no information on negative controls

In conductometry, there is no concept of a "negative control", but factors affecting the conductivity of solutions, such as temperature, are taken into account. When calculating the electrolyte yield, the background value for the distilled water used in the experiment was measured beforehand. This background value was subtracted from the total conductivity measured for the control and experimental samples, as well as for the samples after boiling. All measurements were performed at the same temperature.

4. Are the results clearly presented?
5. a) Inconsistency between the text and tables (e.g., data for 2,4-D in Table 1)

The sentence on lines 191-192 (old version of manuscript) indicates the observed difference. For better understanding, the phrase has been replaced. The phrase «At the same time, inhibition of dry root biomass accumulation under the action of 1 μM and 10 μM 2,4-D did not exceed 25 and 60%, respectively» was replaced «At the same time, inhibition of dry root biomass accumulation under the action of 1 μM and 10 μM 2,4-D was 25 and 60%, respectively.» (lines 221-222).

1. b) Tables 2 and 3 are very dense – difficult to extract key information

Thank you for your significant comment. We agree that Tables 2 and 3 contain a large amount of data, and we considered dividing each table into smaller tables. However, this approach introduces another problem for the reader: the need to compare data from a large number of tables. Unfortunately, such a division would only increase the complexity of the text. For better understanding, we have highlighted the data for five key fatty acids (palmitic, stearic, oleic, linoleic, and α-linolenic acids) in bold. This may make it easier to find key information.

1. c) No concise summary of the main changes in fatty acids

Thank you very much for pointing out the missing summary. As noted, a short summary has been added after the description of the data presented in the tables in the revised manuscript. Lines 448-451

Overall, analysis of FA content changes in etiolated and green spring wheat seedlings revealed that NAA, 2,4-D, and CLD increased the content of key SFAs and UFAs in etiolated seedlings but decreased their content in green seedlings. CLD had the most pronounced effect on OCFA and VLCFA content.

1. d) Some results are described selectively (omitting inconvenient data) Specific examples:
2. e) Table 1, lines 191–192: authors claim that 2,4-D at 1 μM did not affect root dry mass in green seedlings, but data indicate a 25% reduction

The sentence on lines 191-192 (old version of manuscript) indicates the observed difference. However, for better understanding, the phrase was replaced (see point 4a): «At the same time, inhibition of dry root biomass accumulation under the action of 1 μM and 10 μM 2,4-D was 25 and 60%, respectively».

1. f) Inconsistency in the description of CLD’s effect – authors state it does not influence growth, yet data show effect on cell membrane permeability

Indeed, experiments have shown differences in the effects of СLD on the growth parameters of spring wheat seedlings and on membrane permeability. We highlight these differences in section 2.3. « The results indicate that while CLD had no detectable effects on growth, it significantly altered cell membrane structure and permeability». (lines 241-242)

Also, a discussion of the possible influence of CLD on membrane permeability is presented in the Discussion section. « At the same time, the phenolic ring of 2,4-D and the pyridine ring of CLD are much closer in volume to that of natural IAA, thereby allowing them to interact more easily with lipid molecules. In addition, the CLD molecule is capable of forming hydrogen bonds, which also facilitates its interaction with the polar headgroups of phospho- and glycolipid molecules [50].» Lines 553-558.

1. g) No explanation for why CLD has a weaker effect on growth but a stronger effect on cell membranes

Our data on the effect of СLD on growth are consistent with data of other researchers who also found no inhibitory effect of СLD on root and shoot growth in winter wheat (Pyone et al. 2025, https://doi.org/10.1371/ journal.pone.0330225). However, the change in membrane permeability may be due to changes in the FA composition of the membranes under the influence of CLD, as well as the direct interaction of this compound with lipid molecules. These considerations are presented in the discussion section (lines 540-558).

1. h) Assessment of toxicity should be based on observed experimental effects, not assumptions

In our work, we tested toxicity based on the ability of synthetic auxins (NAA, 2,4-D, and CLD) to disrupt physiological functions and metabolism. In describing our results, we highlight the observed experimental effects (changes in growth parameters, electrolyte yield, changes in pigment content, lipid peroxidation products, and fatty acids). To provide a complete picture of the physiological and biochemical changes, we draw on literature data and discuss possible causes of the observed effects. The hypotheses we introduce in this discussion serve primarily as guidelines for future research.

1. i) Line 298: “Treatment with 1 μM NAA did not affect UFA content” – data show a trend

Yes, we agree that there is a slight tendency for the content of individual UFAs (in particular, linoleic and α-linolenic acids) to increase under the influence of 1 μM NAA, but no statistically significant differences were found, so we do not mention them in order not to confuse the readers.

1. j) Inconsistent reporting of CLD effects – sometimes described as “weak,” sometimes as “significant”

Thank you very much for your valuable comment. When describing the weak effects of CLD, we primarily meant its weak influence on seedling growth parameters. We corrected the manuscript accordingly and clarified that the weak effects of CLD are related to its effect on growth.

Concentration selection and limitations:

1. k) Observed effects may be specific to the wheat cultivar used

We thank the reviewer for the important comment. The following phrase has been added to the conclusion. «As the obtained results may be cultivar-specific, further research on various winter and spring wheat cultivars is required to identify general patterns».

5. Are the conclusions supported by the results?
6. a) Mechanisms of PUFA reduction (lines 577–593) – speculation on desaturase activity without direct measurements

In the discussion of the results, we consider possible reasons for the decrease in PUFAs and suggest a decrease in desaturase activity as one of the possible reasons: “The decrease observed in the content of PUFAs, especially linoleic and α-linolenic acids, in green seedlings exposed to NAA, 2,4-D, and CLD may be attributed to several mechanisms: reduced synthesis of FA precursors (e.g., stearic acid C18:0), increased lipid peroxidation (LPO), and decreased desaturase activity.” One of the most frequently used methods for assessing desaturase activity is the calculation of desaturase activity from the ratio of the corresponding UFAs, which are the products of the desaturase reaction, to the SFAs and UFAs, which are the substrates of this reaction (Pamplona et al., 1998, Journal of Lipid Research, Volume 39, Issue 10, 1989 – 1994; De Castro et al., 2012, https://doi.org/10.1590/S1415-52732012000100005). Such calculations are performed very often, even when the content of individual FAs is presented as a weight percentage of the total FA content. In our work, we determined the absolute content of individual fatty acids in the sample, which makes our assumptions regarding desaturase activity quite reasonable.

1. b) “NAA primarily disrupted PUFA biosynthesis” – no direct enzymatic evidence

We agree with the comment, we changed the phrase in the supposed context «In green seedlings, NAA disrupted PUFA biosynthesis probably by inhibiting the synthesis and elongation of SFAs, which serve as essential precursors for PUFA formation”.

1. c) Conclusions about membrane interactions (lines 509–523) – mainly based on chemical structure, not experiments

We do not draw conclusions about membrane interactions, but rather suggest these interactions based on literature data; the phrase is constructed in a hypothetical manner: “We propose that the increased efflux of electrolytes observed under the influence of 2,4-D and CLD arises not from biochemical modification of membrane lipid composition, but instead results from direct interaction of these molecules with polar lipids.”

Logical issue:

1. d) Authors claim CLD has “weak physiological effects” but simultaneously show significant biochemical changes – internally contradictory

We have corrected «weak physiological effects of CLD» to «weak effects of CLD on growth». In the text, we draw readers' attention to the fact that a lack of substance effect on growth does not guarantee the lack of substance effect on biochemical parameters.

6. Are all figures and tables clear and well-presented?
7. a) Figure 1: error in caption (2,4-D ≠ α-naphthylacetic acid); chemical structure should be labeled correctly

We sincerely thank the reviewer for the attention. The error in the figure caption has been corrected. The corresponding corrections have been made to the text (Figure 1). «…NAA, (1-naphthyleneacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid) and CLD (3,6-dichloro-2-pyridinecarboxylic acid, clopyralid)».

1. b) Figure 2: Photos informative, but difficult to assess differences without scale bars; notation “1µM” should be “1 µM”

We thank the reviewer for the comment. We have provided photographs with a ruler and scale bar and have added spaces marked "1 µM."

1. c) Figure 3: Statistical labels inconsistent; Y-axis scale could be better adjusted; numeric values on bars missing

The vertical axes in the upper and lower parts of the figures have been adjusted, and the major axis divisions are labeled. The corrected figures have been added to the manuscript.

1. d) Figure 5: Three panels with different scales; error bars should be consistent, The error bars should be checked, they probably should be the same in the up and down directions,

We thank the reviewer for the useful comment. The vertical axes have been adjusted, and new figures are included in the manuscript. Figure 5 presents the means and standard deviations. Error bars are plotted equally above and below the mean.

1. e) Figure 6: Similar issues as Figure 5; The error bars should be checked, they probably should be the same in the up and down directions,

The median (Q₅₀) is presented in Figure 6 because the LPO product content data showed a non-normal distribution. The error bars indicate the spread of values between the 25^th and 75^th quartiles (Q₂₅ and Q₇₅). Because the data distribution differs from normal, the values between quartiles are also trend toward one side or the other. Therefore, the error bars indicate this alteration and are distributed unevenly around the median.

1. f) In Table 1 (lines 169–177), some values share the same letters (e.g., a, b) but differ significantly.

We thank the reviewer for the comment. Indeed, at first glance, the data on the effects of 1 μM 2,4-D and 1 and 10 μM CLD, for example, on the accumulation of dry mass in etiolated seedlings, would seem to differ. Unfortunately, however, statistical analysis of the data showed that these differences cannot be considered significant at p < 0.05.

1. g) Tables 2 and 3: Too complex, errors “ΣTSFA” instead of “ΣTFA”; symbol “●” for palmitoleic acid used inconsistently; suggest splitting or adding summary tables General issues in data presentation:

We thank the reviewer for the important suggestion. To facilitate the comprehension of the data presented in the tables, we have highlighted the data for 5 key fatty acids (palmitic, stearic, oleic, linoleic, and α-linolenic acids). This will make it easier to find key information. The typo "Σ_TSFA" in the caption to Table 3 has been corrected to "Σ_TFA". The ● sign was introduced to draw the reader's attention to the fact that by C_16:1 we mean not only palmitoleic acid but also other isomers of hexadecenoic acid. For better understanding, the phrase has been changed to "sum of isomers of hexadecenoic acid (including palmitooleic acid)".

1. h) Inconsistent use of commas and dots as decimal separators

The reviewer probably meant the designation 2,4-D, we have corrected periods to commas throughout the text, in tables and figures.

1. i) Lines 44–45 and 56: NAA incorrectly referred to as “α-naphthylacetic acid”; correct: “1naphthaleneacetic acid”

According to the comment, «α-naphthylacetic acid» has been replaced to «1-naphthaleneacetic acid».

1. j) Line 58: “Despite being in use for nearly a century” – 2,4-D was introduced in the 1940s, so less than 100 years

According to the comment, the phrase has been changed: «Despite being in use for more than 80 years…».

7. Additional mistakes
8. a) Line 58: “Despite being in use for nearly a century” – 2,4-D was introduced in the 1940s, so it has been in use for less than 100 years.

According to the comment, the phrase has been changed: «Despite being in use for more than 80 years…».

1. b) Line 436: mistake in reference numbering “[36, 27]” – it should be “[26, 27]”.

Due to changes in text and addition of references the numbering has been changed.

2. c) Table 1 header: “2.4-D” should be “2,4-D” (dot replaced with comma).

Thank you for the comment, we have corrected it.

We would like to thank you again for your valuable time and important comments, which allowed us to carry out the necessary work to correct and improve the manuscript. Your suggestion has been extremely helpful to us.

With best regards, authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting paper that is technically sound.   It can have more impact if some of the results and discussion is expanded to include recent work.

Specific Comments:

• The comparison between etiolated and green seedlings provides a comprehensive view of how synthetic auxins impact wheat at different developmental stages. The discussion of the link between reduced PUFAs and impaired photosynthesis needs to be expanded in light of other recent work.
• The use of chromatography-mass spectrometry to assess fatty acid profiles lends strong quantitative support to the conclusions. Please add a discussion on distinguishing between NAA, 2,4-D, and CLD.
• How does this work directly impact on modern agriculture?
• The auxin concentrations in the auxin experiment reflect typical residual levels found in agricultural soils after herbicide application. How do these levels correlate with real-world pesticide concentrations?
• Can the authors expand the discussion of the differential effect on fatty acid content between etiolated (increased) and green (decreased) seedlings?
• The link between lipid peroxidation for CLD can be made more explicit.
• Can the authors explain any Structure-activity relationships for plant metabolism of the molecules in this study? This can improve the conclusion and discussion sections.
• What would be the effects of applying specific micronutrients on mitigating the toxic effects on the photosynthetic apparatus and fatty acid metabolism in wheat seedlings?

 

Author Response

Dear reviewer,

We sincerely thank you for the positive assessment of our work and for recognizing its relevance and scientific value. We appreciate the constructive recommendations aimed at improving the quality of discussion and manuscript's presentation. All suggested improvements have been carefully implemented.

Additions to the text are highlighted in green.

Below are responses to comments.

• The comparison between etiolated and green seedlings provides a comprehensive view of how synthetic auxins impact wheat at different developmental stages. The discussion of the link between reduced PUFAs and impaired photosynthesis needs to be expanded in light of other recent work.

Thanks to the reviewer for the valuable comment.

We incorporated the detailed discussion of this question. Lines 626-644.

Various experimental models and plant species have demonstrated a link between PUFA content and photosynthesis. On one hand, high photosynthetic activity is a necessary factor for PUFA synthesis in green plant tissues. For example, when comparing two tea cultivars, Jincha 2 and Wuniuzao, higher levels of FA synthesis precursors, α-linolenic acid, and its derivatives (such as methyl dihydrojasmonate) were found in Jincha 2, which exhibited higher photosynthetic rates, stomatal conductance, and transpiration rates [83]. Conversely, high PUFA content in thylakoid membrane lipids makes them susceptible targets for ROS and contributes to photosynthesis disruption under stress conditions. This is illustrated by heat-tolerant rice cultivars, which exhibited reduced linoleic and α-linolenic acid content alongside lower electrolyte leakage, reduced malondialdehyde (MDA) accumulation, and maintenance of maximum PSII quantum yield (Fv/Fm) [84]. Similarly, the FA composition of thylakoid membrane lipids shifted toward higher proportions of SFAs and MUFAs in the brown macroalga Undaria pinnatifida following prolonged high-intensity light exposure [85]. Increased SFA content has also been observed in Amaranthus caudatus and wheat (T. aestivum) under drought conditions [86]. Thus, reduced PUFA content is not merely a consequence of impaired photosynthetic activity but also contributes to membrane stabilization by limiting lipid peroxidation intensity.

• The use of chromatography-mass spectrometry to assess fatty acid profiles lends strong quantitative support to the conclusions. Please add a discussion on distinguishing between NAA, 2,4-D, and CLD.

We incorporated the discussion of the differences between NAA, 2,4-D and CLD.

Lines  593-599.

Notably, in etiolated seedlings, treatment with 10 μM CLD resulted in the greatest increase in key SFAs (palmitic and stearic acids) and MUFA (oleic acid). Concurrently, VLCFA content decreased to undetectable levels (Table 2). These data suggest that CLD may inhibit VLCFA elongases, similar to acetamide, chloroacetanilide, and oxyacetamide herbicides [43,44]. However, given the limited research on this mechanism, this hypothesis remains speculative and requires further investigation.

Lines 662-671.

If we assume that CLD reduces UFA content by activating LPO processes, it is noteworthy that in most cases this reduction was observed at 1 μM, but not at 10 μM CLD (Table 3). ). This differs significantly from the results obtained for NAA and 2,4-D, which more markedly reduced the UFA content in green seedling shoots at 10 μM (Table 3). Auxin-type herbicides are known to enhance the generation of ROS in plant tissues, which affects the activity of antioxidant defence enzymes in cells [87]. We suggest that low CLD concentrations were insufficient for rapid activation of this defence system, but that an increased dose of CLD enhanced the activity of the antioxidant enzymes. Our preliminary experimental data suggest this particular mechanism, but further verification is required.

 

• How does this work directly impact on modern agriculture?

Thanks to the reviewer for their valuable comments. The corresponding corrections have been made in the Conclusion: «The data obtained in this study highlight the need for monitoring herbicides residues in soils and assessing their risks to the development and physiological status of major crops, including cereals. As the obtained results may be cultivar-specific, further research on various winter and spring wheat cultivars is required to identify general patterns». Lines 829-833.

• The auxin concentrations in the auxin experiment reflect typical residual levels found in agricultural soils after herbicide application. How do these levels correlate with real-world pesticide concentrations?

Thanks to the reviewer for an interesting and important question. If we calculate the recommended treatment regimes for spring wheat crops, for example, for CLD, a component of Hacker 300 SL (soluble liquid), and 2,4-D, a component of Chistalan, manufactured by JSC August Inc. (Moscow, Russia), at a treatment rate of 300-400 L/ha and an active ingredient content of 300 and 376 g/L, respectively, this corresponds to approximately 10-15 g of active ingredient per m². This treatment results in a significantly higher concentration of herbicides per plant than we used in our study. Typically, treatment with these products is carried out at the tillering stage, when the cereals have developed their integumentary and mechanical tissues. Thus, cereal resistance is largely due to the anatomical and morphological features of leaf structure (high wax content and a nearly vertical arrangement), which impede herbicide absorption. Therefore, in our study, we focused primarily on residual herbicide concentrations found in water and soil, as well as slight excesses of the maximum permissible concentration. In preliminary experiments, analyzing the impact on the growth characteristics of winter (Irkutskaya variety) and spring wheat (Novosibirskaya 29 variety), we found that herbicide concentrations exceeding 10 μM had an excessively strong effect on seedlings. This was particularly evident when treating seedlings with 2,4-D, which at high concentrations led to serious disruptions in seedling development, specifically the formation of calluses instead of roots.

• Can the authors expand the discussion of the differential effect on fatty acid content between etiolated (increased) and green (decreased) seedlings?

We thank the reviewer for their important contribution. The necessary corrections have been made to the text. Lines 571-581.

A similar increase in PUFA content in heterotrophic tissues was observed in soybean zygotic embryos treated with exogenous auxins (NAA and 2,4-D) [71]. Of particular interest, NAA (10 mg/L) specifically increased the level of linoleic acid but not α-linolenic acid, a result that aligns with our findings (Table 2). The authors conclude that 2,4-D enhanced the efficiency of desaturation processes in extraplastidial compartments. Conversely, in green seedlings, a significant decrease in FA content was observed, particularly affecting PUFAs, specifically linoleic and α-linolenic acids (Table 3). Currently, no data indicate that 2,4-D or other auxin-type herbicides directly affect the activity of enzymes involved in fatty acid synthesis in plant green tissues. Instead, the reduction in specific FAs caused by these compounds is primarily attributed to oxidative stress [21,72].

• The link between lipid peroxidation for CLD can be made more explicit.

We thank the reviewer for their valuable comment. The following discussion has been added to the text. Lines 662-671.

If we assume that CLD reduces UFA content by activating LPO processes, it is noteworthy that in most cases this reduction was observed at 1 μM, but not at 10 μM CLD (Table 3). ). This differs significantly from the results obtained for NAA and 2,4-D, which more markedly reduced the UFA content in green seedling shoots at 10 μM (Table 3). Auxin-type herbicides are known to enhance the generation of ROS in plant tissues, which affects the activity of antioxidant defence enzymes in cells [87]. We suggest that low CLD concentrations were insufficient for rapid activation of this defence system, but that an increased dose of CLD enhanced the activity of the antioxidant enzymes. Our preliminary experimental data suggest this particular mechanism, but further verification is required.

• Can the authors explain any Structure-activity relationships for plant metabolism of the molecules in this study? This can improve the conclusion and discussion sections.

In the discussion this explanation is given. Lines 540-558.

The minor effects of CLD on wheat seedling growth suggest negligible biochemical and cellular transformations when exposed to this compound. Nonetheless, we have identified multiple CLD-induced changes in FA content (Tables 2 and 3), which were not always accompanied by changes in membrane permeability (Figure 4). Specifically, the application of NAA caused marked changes in seedling growth and essential FAs levels, yet no changes in membrane permeability were observed (Tables 2 and 3; Figure 2-4). We propose that the increased efflux of electrolytes observed under the influence of 2,4-D and CLD arises not from biochemical modification of membrane lipid composition, but instead results from direct interaction of these molecules with polar lipids. Indeed, the chemical structure of 2,4-D, particularly that of CLD, appears to facilitate such interactions. As previously reported by M. Flasiński and K. Hąc-Wydro, natural IAA binds more strongly to lipid monolayers than does NAA [68]. Since the naphthalene ring of the NAA molecule occupies a much larger volume than the indole system of IAA, its penetration between the polar headgroups of phospholipids becomes restricted [68]. At the same time, the phenolic ring of 2,4-D and the pyridine ring of CLD are much closer in volume to that of natural IAA, thereby allowing them to interact more easily with lipid molecules. In addition, the CLD molecule is capable of forming hydrogen bonds, which also facilitates its interaction with the polar headgroups of phospho- and glycolipid molecules [50].

• What would be the effects of applying specific micronutrients on mitigating the toxic effects on the photosynthetic apparatus and fatty acid metabolism in wheat seedlings?

We are grateful to the reviewer for a very interesting question. In general, the addition of micronutrients has multidirectional effects, depending on the target and the nature of the micronutrient. For example, lower Zn levels were shown to have a beneficial effect on the levels of protein, fatty acids, and photosynthetic pigments in the microalgae Dunaliella tertiolecta (El-Agawany and Kaamoush, 2023, https://doi.org/10.1007/s11356-022-20536-z). Increased uptake of Ba, Cr, Mn, Ni, and Mo, as well as the synthesis of PUFAs, were observed in the aquatic plant Salvinia natans, when supplemented with 1 μg/L Li to the medium, although no significant changes in chlorophyll content were detected (Török et al., 2023, https://doi.org/10.3390/molecules28145347). Treatment of marjoram with Fe, Zn, and Mn increased the content of chlorophylls and carotenoids and also promoted an increase in the synthesis of essential oil in plants (El-Khateb et al., 2020, Plant Archives, Volume 20 No. 2,  pp. 8315-8324). In durum and bread wheat, excess B has been shown to negatively impact Ca levels, which may be important for plant productivity (Vera-Maldonado et al., 2024, https://doi.org/10.3389/fpls.2024.1332459). Studies analyzing the effects of exogenous auxins on plant physiological status and micro- and macronutrient metabolism indicate that high doses of herbicides promote the uptake and accumulation of most metals in roots (Skiba et al., 2017, http://dx.doi.org/10.1016/j.envpol.2016.10.072). There is also evidence that the addition of exogenous auxin (IAA) to the nutrient medium led to a decrease in the level of UFAs in spring wheat shoot tissue, and the addition of NaNO₃ to the medium somewhat mitigated this effect (Kovalevskaya, 2023, https://doi.org/10.31857/S0233475522060081 (in Russian)). Based on our preliminary results from experiments examining the effects of exogenous auxins on spring wheat seedling development, we can conclude that their negative impact (especially 2,4-D) persisted after transferring the plants to ½ Knop’s solution and discontinuing auxin treatment. However, this issue requires further detailed research.

Since this manuscript is quite lengthy, and studying the effects of micronutrients was not the goal of our work, we have not included our discussion of this topic in the manuscript.

We would like to thank you again for your valuable time and important comments, which allowed us to carry out the necessary work to correct and improve the manuscript. Your suggestion has been extremely helpful to us.

With best regards, authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

I ultimately consider that the revision meets my expectations.

The only remaining issue is checking the comma between 2.4-D, whether it should ultimately be a comma or a period.
The second matter concerns the spacing between, for example, 1 and a micromole. In Fig. 2, this has not been done. I leave this to the Editors’ decision.
In the abbreviations (line 823), there is still an error: “NAA α-Naphthylacetic acid” instead of “1-naphthaleneacetic acid.”