Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the ms Detection of plant hormones in stigma exudates and their role in pollen germination-Horticulturae-3822282

This study explores the hormonal composition of stigma exudates in wet-stigma plants and their role in pollen germination and ROS balance. Using UPLC-ESI-MS profiling with in vitro germination assays, it addresses an underexplored topic in plant reproductive biology.

I have only some comments to improve the ms:

1. The text should be carefully proofread, as it contains numerous typographical errors, most notably missing spaces.
2. I suggest including the two main hormones, JA and ABA, in the title, as the study focuses on their effects.
3. In the Introduction, please elaborate on the hypothesis (based on the literature and knowledge), and clearly state which experiments and methods were chosen to test it.
4. I suggest presenting the structure of the flowers of the species under study, with particular attention to the characteristics of the stigma.
5. Abscisic acid (ABA) is well known to inhibit germination at high concentrations. Is there a relationship between pollen and seed germination in response to ABA? I suggest interpreting this in light of the results. I suggest that authors include a separate section on this topic, incorporating studies that have investigated ABA-mediated inhibition of germination, for example Kutasy et al., 2025 Garlic Extracts Nanoliposome as an Enhancer of Bioavailability of ABA and Thiamine Content and as an Antifungal Agent Against Fusarium oxysporum f. sp. pisi Infecting Pisum sativum https://doi.org/10.3390/agronomy15040991

6. Table1 The unit of measurement should be included in the table.
7. I suggest that the authors include in the discussion their predictions for the practical applications of these results.

After minor revisions, I consider the manuscript suitable for publication.

Author Response

We thank the esteemed Reviewer for his careful attention to our manuscript. 

1. The text should be carefully proofread, as it contains numerous typographical errors, most notably missing spaces.

The three of us carefully proofread the text twice and found typos and errors.

2. I suggest including the two main hormones, JA and ABA, in the title, as the study focuses on their effects.

We have changed the title according to your recommendation.

3. In the Introduction, please elaborate on the hypothesis (based on the literature and knowledge), and clearly state which experiments and methods were chosen to test it.

We have formulated the hypothesis and included our methods in the final paragraph of the Introduction.

4. I suggest presenting the structure of the flowers of the species under study, with particular attention to the characteristics of the stigma.

All plants included in this study have a fairly typical pistil structure: a narrow style ends in a widening - a stigma, the area of ​​which we measured, and on it was located a drop of exudate that we collected. Photos of flowers are shown in Figure 1, with scale sections so that you can estimate the size.

5. Abscisic acid (ABA) is well known to inhibit germination at high concentrations. Is there a relationship between pollen and seed germination in response to ABA? I suggest interpreting this in light of the results. I suggest that authors include a separate section on this topic, incorporating studies that have investigated ABA-mediated inhibition of germination, for example Kutasy et al., 2025 Garlic Extracts Nanoliposome as an Enhancer of Bioavailability of ABA and Thiamine Content and as an Antifungal Agent Against Fusarium oxysporum f. sp. pisi Infecting Pisum sativum https://doi.org/10.3390/agronomy15040991

 We have included in the Discussion a paragraph devoted to the inhibitory properties of ABA, including a link to the cited publication and other articles on the topic.

6. Table1 The unit of measurement should be included in the table.

We have included units of measurement in Table 1. All values ​​were converted to stigma area, since otherwise it would not be possible to compare flowers of different sizes.

7. I suggest that the authors include in the discussion their predictions for the practical applications of these results.

For now, our research is of a fundamental nature, however, as soon as we move into the practical realm, and there is such a plan, we will definitely refer to this article.

Reviewer 2 Report

Comments and Suggestions for Authors

While the topic of plant hormones in stigma exudates and their role in pollen germination is of considerable interest, the manuscript in its current form suffers from fundamental limitations regarding plant materials, data quality, and analytical approaches.  Before this work can undergo further professional review, the authors must address at least the following critical issues:
--The objectives and significance of this study should be clearly stated.
--The genetic background of the plant materials must be clearly specified.
--There are apparent errors in the statistical significance analysis that require correction.
--The Results section contains inconsistencies in plant species/context.

Author Response

We are grateful to the esteemed reviewer for his critical attitude and valuable comments.

1. We have added a paragraph to the Introduction formulating the hypothesis, objectives and methods.
2. As for the plants, tobacco Petit Havana belongs to pure wild-type lines, no genetic changes have been made to these plants, they have been used in research for several decades (Breygina et al. 2009, 2012, 2022; Schekaleva et al. 2024), self-pollinated and grown from seeds every year. The Samsun tobacco variety was provided to us by colleagues from the All-Russian Research Institute of Plant Breeding, it also represents a laboratory pure wild-type line, on the basis of which colleagues obtain various genetic modifications described in other works (Raldugina et al. 2021; Podobedova et al. 2025). We cannot describe Streptocarpus in a genetic sense, since it is not a laboratory line - we could not find a place where these plants would be grown for research, so we bought several seedlings in a plant store. The remaining plants were purchased by Botanical Gardens for the collection and were not modified in the laboratory. We have provided all available information in this paper, as well as in the previous article, carried out on these plants, which was published in the journal Plant Growth Regulation (Breygina et al. 2025).
3. We checked the statistical criteria, everywhere we correctly indicated the reliability. In addition, we added another criterion for mass samples, which we used to calculate new data, for example, on the fluorescence of calcium-dependent dye.
4. We have significantly modified the Results, adding new data and improving the text.

Breygina M, Luneva O, Babushkina K, Kushunina M, Lazareva N, Klimenko E (2025) Which reactive oxygen species are produced on wet stigmas of different monocot plants? Plant Growth Regulation. doi:10.1007/s10725-025-01330-6.

Breygina MA, Matveeva NP, Ermakov IP (2009) The role of Cl¯ in pollen germination and tube growth. Russian Journal of Developmental Biology 40, 157–164. doi:10.1134/S1062360409030047.

Breygina M, Matveyeva N, Polevova S, Meychik N, Nikolaeva Y, Mamaeva A, Yermakov I (2012) Ni²⁺effects on Nicotiana tabacum L. pollen germination and pollen tube growth. BioMetals 25,. doi:10.1007/s10534-012-9584-0.

Breygina M, Schekaleva O, Klimenko E, Luneva O (2022) The balance between different ROS on tobacco stigma during flowering and its role in pollen germination. Plants 11, 993.

Podobedova A, Baranova EN, Gulevich AA, Chaban IA, Breygina M (2025) Comprehensive Study of Sexual Reproduction in Nicotiana tabacum Plants Overexpressing H2O2-Producing Enzymes: Superoxide Dismutase and Choline Oxidase. Plants 14, 2103. doi:10.3390/plants14142103.

Raldugina GN, Evsukov S V, Bogoutdinova LR, Gulevich AA, Baranova EN (2021) Morpho-physiological testing of NaCl sensitivity of tobacco plants overexpressing choline oxidase gene. Plants 10,. doi:10.3390/plants10061102.

Schekaleva O, Luneva O, Klimenko E, Shaliukhina S, Breygina M (2024) Dynamics of ROS production, SOD, POD and CAT activity during stigma maturation and pollination in Nicotiana tabacum and Lilium longiflorum. Plant Biology. doi:10.1111/plb.13677.

Reviewer 3 Report

Comments and Suggestions for Authors In this manuscript, the authors detected the phytohormones in stigma exudates of several plant species with wet stigmas. The results help in understanding the role of stigma exudates in pollination. The finding that JA and ABA are present in stigma exudates and may possibly promote pollen germination is novel and interesting. However, the current data are still insufficient to draw firm conclusions. More detailed information should be provided before it can be accepted. Major points 1. The authors showed the results of added JA, MeJA, and IleJA on pollen germination of tobacco in Figures 2 and 3. However, they only showed the effect of JA on pollen germination of Streptocarpus in Figure 3B. In my opinion, the effects of MeJA and IleJA on pollen germination of Streptocarpus should also be provided. To compare the dose-dependence of JA, MeJA, and IleJA in promoting pollen germination of both species, the same concentration series should be used. 2. In Figure 4, the authors showed the effect of adding 10 μM ABA on pollen germination in five plant species. In Figure 5, they demonstrated the effect of ABA on H2O2 accumulation in tobacco pollen grains. The effect of added ABA on tobacco pollen germination should also be included. Additionally, the dose-dependent effects of ABA on pollen germination across different plant species should be presented. 3. In Figure 5, the effect of serial concentrations of JA and ABA on H2O2 accumulation in pollen grains should be shown. 4. To illustrate the mechanism of ABA and JA promotion on pollen germination, simply detecting their effect on ROS accumulation is not enough. The effect of the two hormones on additional cytoplasmic parameters, such as Ca2+, should also be examined. Minor points 1. In Figure 1, the authors provided photographs of several plant species. In my opinion, the photographs should be moved to the M&M section, as these photos should not be presented as experimental data. 2. The text contains some formatting errors that need to be reviewed and corrected.

Author Response

We thank the esteemed Reviewer for valuable comments that helped to significantly improve the manuscript!

 

1. The authors showed the results of added JA, MeJA, and IleJA on pollen germination of tobacco in Figures 2 and 3. However, they only showed the effect of JA on pollen germination of Streptocarpus in Figure 3B. In my opinion, the effects of MeJA and IleJA on pollen germination of Streptocarpus should also be provided. To compare the dose-dependence of JA, MeJA, and IleJA in promoting pollen germination of both species, the same concentration series should be used.

We have conducted many other experiments (see below), but streptocarpus pollen does not store well, it only germinates when fresh. Unfortunately, this summer the streptocarpus finished flowering at the end of June, and we were unable to conduct experiments. If we had bought other plants, it would have been non-standard, since we had previously conducted all experiments with these plants of a certain variety.

2. In Figure 4, the authors showed the effect of adding 10 μM ABA on pollen germination in five plant species. In Figure 5, they demonstrated the effect of ABA on H2O2 accumulation in tobacco pollen grains. The effect of added ABA on tobacco pollen germination should also be included. Additionally, the dose-dependent effects of ABA on pollen germination across different plant species should be presented.

We previously conducted an experiment on the dependence of tobacco pollen germination on the concentration of ABA. These data were published in the journal Biomolecules in 2023. To add tobacco to the range of other species affected by ABA, we added another concentration of 10 μM here. Figure 4 has been modified - now tobacco is also added, and this is logical.

3. In Figure 5, the effect of serial concentrations of JA and ABA on H2O2 accumulation in pollen grains should be shown.

We have conducted such a series of experiments and obtained very important data that lower concentrations of hormones cause a moderate increase in the concentration of peroxide. This is very interesting! We thank you for this idea. The data are added to Results, Figure 5.

4. To illustrate the mechanism of ABA and JA promotion on pollen germination, simply detecting their effect on ROS accumulation is not enough. The effect of the two hormones on additional cytoplasmic parameters, such as Ca2+, should also be examined.

Thank you very much for this idea, we did an additional experiment and found that intracellular calcium does not change under the influence of 10 μM jasmonic acid and ABA. At the same time, an antioxidant effect is observed in pollen. Apparently, it is not associated with calcium signaling, although I admit that we simply could not see short and rapid changes in calcium. In any case, if the signaling mechanism was the same as in guard cells, we would have seen an increase in calcium. Thus, the signaling mechanism in pollen grains is different from that realized in some other specialized cells. This result made the work more complete. The data are added to Results, Table 2.

Minor points 1. In Figure 1, the authors provided photographs of several plant species. In my opinion, the photographs should be moved to the M&M section, as these photos should not be presented as experimental data. 2. The text contains some formatting errors that need to be reviewed and corrected.

We have moved Figure 1 to Materials and Methods. The text was carefully proofread and double-checked.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author did not address all the questions raised, particularly Question 1, Question 3, and Question 4. Instead, a list of references was provided at the end without any substantive explanation. Furthermore, no substantial revisions were made to the text, with only minor changes implemented. At this stage, it is recommended that the manuscript not be published.

Author Response

We regret that we were unable to satisfy the requests of the esteemed Reviewer, as they were not stated specifically and clearly.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors has answered all my questions properly. The revised manuscript is acceptable.

Author Response

We thank the esteemed Reviewer for the positive assessment of our manuscript and the work done to improve it.